Devices, systems, and methods for the fabrication of tissue

ABSTRACT

Described herein are bioprinters comprising: one or more printer heads, wherein a printer head comprises a means for receiving and holding at least one cartridge, and wherein said cartridge comprises contents selected from one or more of: bio-ink and support material; a means for calibrating the position of at least one cartridge; and a means for dispensing the contents of at least one cartridge. Further described herein are methods for fabricating a tissue construct, comprising: a computer module receiving input of a visual representation of a desired tissue construct; a computer module generating a series of commands, wherein the commands are based on the visual representation and are readable by a bioprinter; a computer module providing the series of commands to a bioprinter; and the bioprinter depositing bio-ink and support material according to the commands to form a construct with a defined geometry.

CROSS-REFERENCE

This application claims the benefit of U.S. provisional application No.61/405,582, filed on Oct. 21, 2010, and is a continuation of U.S.application Ser. No. 13/246,428, filed Sep. 27, 2011, each of which isincorporated by reference in its entirety.

BACKGROUND OF INVENTION

A number of pressing problems confront the healthcare industry. As ofDecember 2009 there were 105,305 patients registered by United Networkfor Organ Sharing (UNOS) as needing an organ transplant. Between Januaryand September 2009, only 21,423 transplants were performed. Each yearmore patients are added to the UNOS list than transplants are performed,resulting in a net increase in the number of patients waiting for atransplant. For example, at the beginning of 2008, 75,834 people wereregistered as needing a kidney; at the end of that year, the number hadgrown to 80,972. 16,546 kidney transplants were performed that year, but33,005 new patients were added to the list. The 2008 transplant rate fora patient registered by UNOS as needing a kidney was 20%. The mortalityrate of waitlist patients was 7%.

Additionally, the research and development cost of a new pharmaceuticalcompound is approximately $1.8 billion. See Paul, et al. (2010). How toimprove R&D productivity: the pharmaceutical industry's grand challenge.Nature Reviews Drug Discovery 9(3):203-214. Drug discovery is theprocess by which drugs are discovered and/or designed. The process ofdrug discovery generally involves at least the steps of: identificationof candidates, synthesis, characterization, screening, and assays fortherapeutic efficacy. Despite advances in technology and understandingof biological systems, drug discovery is still a lengthy, expensive, andinefficient process with low rate of new therapeutic discovery.

SUMMARY OF THE INVENTION

There is a need for tools and techniques that facilitate application ofregenerative medicine and tissue engineering technologies to relievingthe urgent need for tissues and organs. There is also a need for toolsand techniques that substantially increase the number and quality ofinnovative, cost-effective new medicines, without incurringunsustainable R&D costs. Accordingly, the inventors describe hereindevices, systems, and methods for fabricating tissues and organs.

Described herein are bioprinters comprising: one or more printer heads,wherein a printer head comprises a means for receiving and holding atleast one cartridge, and wherein said cartridge comprises contentsselected from one or more of: bio-ink and support material; a means forcalibrating the position of at least one cartridge; and a means fordispensing the contents of at least one cartridge. In one embodiment, aprinter head described herein comprises a means for receiving andholding one cartridge. In another embodiment, a printer head describedherein comprises a means for receiving and holding more than onecartridge. In another embodiment, the bioprinter further comprises aprinter stage. In another embodiment, the means for receiving andholding at least one cartridge is selected from: magnetic attraction, acollet chuck grip, a ferrule, a nut, a barrel adaptor, or a combinationthereof. In yet another embodiment, the means for receiving and holdingat least one cartridge is a collet chuck grip. In yet anotherembodiment, the means for calibrating the position of at least onecartridge of is selected from: laser alignment, optical alignment,mechanical alignment, piezoelectric alignment, magnetic alignment,electrical field or capacitance alignment, ultrasound alignment, or acombination thereof. In yet another embodiment, the means forcalibrating the position of at least one cartridge is laser alignment.In another embodiment, the means for dispensing the contents of at leastone cartridge is application of a piston, application of pressure,application of compressed gas, hydraulics, or a combination thereof. Inyet another embodiment, the means for dispensing the contents of atleast one cartridge is application of a piston. In yet anotherembodiment, the diameter of the piston is less than the diameter of acartridge. In another embodiment, the bioprinter further comprises ameans for adjusting temperature. In yet another embodiment, thebioprinter further comprises a means for adjusting the ambienttemperature, the temperature of a cartridge, the temperature of thecontents of the cartridge, the temperature of the receiving surface, ora combination thereof. In yet another embodiment, the means foradjusting temperature is a heating element. In yet another embodiment,the means for adjusting temperature is a heater. In yet anotherembodiment, the means for adjusting temperature is a radiant heater, aconvection heater, a conductive heater, a fan heater, a heat exchanger,or a combination thereof. In yet another embodiment, the means foradjusting temperature is a cooling element. In yet another embodiment,the means for adjusting temperature is a container of coolant, a chilledliquid, ice, a radiant cooler, a convection cooler, a conductive cooler,a fan cooler, or a combination thereof. In yet another embodiment, thetemperature is adjusted to between about 40 and about 90° C. In yetanother embodiment, the temperature is adjusted to between about 0 andabout 10° C. In another embodiment, a bioprinter disclosed herein,further comprises a means for applying a wetting agent to one or moreof: the printer stage; the receiving surface, the deposition orifice,bio-ink, support material, or the printed construct.

Also disclosed herein are methods of calibrating the position of acartridge comprising a deposition orifice, wherein the cartridge isattached to a bioprinter, comprising: calibrating the position of thecartridge along at least one axis; wherein the axis is selected from thex-axis, y-axis, and z-axis; and wherein each calibration is made by useof a laser. In one embodiment, the methods comprise activating the laserand generating at least one substantially stable and/or substantiallystationary laser beam, and where said laser beam is horizontal to theground. In another embodiment, the methods comprise activating the laserand generating at least one substantially stable and/or substantiallystationary laser beam, and where said laser beam is vertical to theground. In yet another embodiment, each calibration is made by use offirst and a second laser. In yet another embodiment, the first laser isvertical to the ground and the second laser is horizontal to the ground.In another embodiment, calibrating the position of the cartridge alongthe y-axis by use of a horizontal laser comprises: positioning thecartridge so that the cartridge is (i) located in a first y octant and(ii) the dispensing orifice is below the upper threshold of the laserbeam; (a) moving the cartridge towards the laser beam and stopping saidmovement as soon as the laser beam is interrupted by the cartridge,wherein the position at which the laser beam is interrupted by thecartridge is the first y position; (b) re-positioning the cartridge sothat the cartridge is located in the second y octant and the dispensingorifice is below the upper threshold of the laser beam; (c) moving thecartridge towards the laser beam and stopping said movement as soon asthe laser beam is interrupted by the cartridge, wherein the position atwhich the laser beam is interrupted is the second y position; (d) andcalculating the mid-point between the first y position and the second yposition. In another embodiment, calibrating the position of thecartridge along the x-axis by use of a horizontal laser comprises: (a)positioning the cartridge (i) at the mid-point between the first yposition and the second y position, and (ii) outside the sensorthreshold of the laser; (b) moving the cartridge towards the sensorthreshold and stopping said movement as soon as the cartridge contactsthe sensor threshold; (c) wherein the position at which the cartridgecontacts the sensor increased by half the cartridge width is the xposition. In another embodiment, calibrating the position of thecartridge along the z-axis by use of a horizontal laser comprises: (a)positioning the cartridge so that the dispensing orifice is locatedabove the laser beam; (b) moving the cartridge towards the laser beamand stopping said movement as soon as the laser beam is interrupted bythe cartridge, wherein the position at which the laser beam isinterrupted is the z position. In another embodiment, calibrating theposition of the cartridge along the y-axis by use of a vertical lasercomprises: (a) positioning the cartridge so that the cartridge is (i)located in a first y octant and (ii) the dispensing orifice is outsidethe sensor threshold of the laser beam; (b) moving the cartridge towardsthe laser beam and stopping said movement as soon as the laser beam isinterrupted by the cartridge, wherein the position at which the laserbeam is interrupted by the cartridge is the first y position; (c)re-positioning the cartridge so that the cartridge is located in thesecond y octant and the dispensing orifice is outside of the sensorthreshold of the laser beam; (d) moving the cartridge towards the laserbeam and stopping said movement as soon as the laser beam is interruptedby the cartridge, wherein the position at which the laser beam isinterrupted is the second y position; (e) calculating the mid-pointbetween the first y position and the second y position; and (f)optionally, repeating (a)-(e) and averaging calculated mid-points. Inanother embodiment, calibrating the position of the cartridge along thex-axis by use of a vertical laser comprises: (a) positioning thecartridge so that the cartridge is (i) located in a first x octant and(ii) the dispensing orifice is outside the sensor threshold of the laserbeam; (b) moving the cartridge towards the laser beam and stopping saidmovement as soon as the laser beam is interrupted by the cartridge,wherein the position at which the laser beam is interrupted by thecartridge is the first x position; (c) re-positioning the cartridge sothat the cartridge is located in the second x octant and the dispensingorifice is outside of the sensor threshold of the laser beam; (d) movingthe cartridge towards the laser beam and stopping said movement as soonas the laser beam is interrupted by the cartridge, wherein the positionat which the laser beam is interrupted is the second x position; (e)calculating the mid-point between the first x position and the second xposition; and (f) optionally, repeating (a)-(e) and averaging calculatedmid-points. In another embodiment, calibrating the position of thecartridge along the z-axis by use of a vertical laser comprises: (a)positioning the printer head so that the dispensing orifice is locatedabove the laser beam and outside of the laser sensor range threshold;(b) moving the printer head along the z-axis the sensor threshold isreached; wherein, the z-position is the position at which the lasersensor threshold is reached; and optionally, repeating steps (a) and (b)and calculating average z-positions. In another embodiment, calibratingthe position of the cartridge along the z-axis comprises: visuallydetermining the position of the dispensing orifice.

Further described herein are systems for calibrating the position of acartridge comprising a dispensing orifice, wherein the cartridge isattached to a bioprinter, said system comprising: a means forcalibrating the position of the cartridge along at least one axis, andwherein the axis is selected from the y-axis, x-axis, and z-axis. In oneembodiment, the means for calibrating the cartridge is laser alignment,optical alignment, mechanical alignment, piezoelectric alignment,magnetic alignment, electrical field or capacitance alignment,ultrasound alignment, or a combination thereof. In another embodiment,the means for calibrating the cartridge is laser alignment. In yetanother embodiment, the laser alignment means comprises at least onelaser, selected from a horizontal laser and a vertical laser. In yetanother embodiment, the laser alignment means comprises a horizontallaser and a vertical laser. In yet another embodiment, the laseralignment means is accurate to ±40 μm on the vertical axis and ±20 μm onthe horizontal axis.

Further described herein are methods for fabricating tissue constructs,comprising: a computer module receiving input of a visual representationof a desired tissue construct; a computer module generating a series ofcommands, wherein the commands are based on the visual representationand are readable by a bioprinter; a computer module providing the seriesof commands to a bioprinter; and the bioprinter depositing bio-ink andsupport material according to the commands to form a construct with adefined geometry. In some embodiments, a computer module comprises adisplay screen. In further embodiments, a computer module comprises agraphical user interface (GUI). In still further embodiments, a userdefines the content of one or more objects to form a visualrepresentation of a desired tissue construct using a GUI provided by thecomputer module. In one embodiment, the display screen consistsessentially of a grid comprising a plurality of objects of substantiallythe same shape and substantially equal size. In yet another embodiment,each object is in the shape of a circle. In yet another embodiment, theuser defines the content of one or more objects to form a visualrepresentation of a desired tissue construct. In yet another embodiment,the user defined content of an object is selected from bio-ink orsupport material. In further embodiments, the display screen consists ofthree-dimensional rendering(s) that are input by the user electronicallyor manually, whereby the various components of the three-dimensionalrendering can be adjusted in any suitable plane or vector prior toexecuting a bioprinting protocol on the bioprinter.

Further described herein are methods of attaching a cartridge to abioprinter, comprising: (a) inserting the cartridge into a collet chuck,wherein the collet chuck is attached to a printer head of thebioprinter; and (b) adjusting the outer collar of the collet chuck;wherein the inserting and adjusting do not substantially alter theposition of the printer head.

Further described herein are cartridges for use with the bioprintersdescribed herein, comprising at least one orifice, wherein the edges ofthe orifice are smooth or substantially smooth. In one embodiment, thecartridge is a capillary tube or a micropipette. In another embodiment,the cartridge comprises a bio-ink. In yet another embodiment, thecartridge comprises a support material. In yet another embodiment, theorifice is circular or square. In yet another embodiment, the cartridgehas an internal diameter of about 1 μm to about 1000 μm. In yet anotherembodiment, the cartridge has an internal diameter of about 500 μm. Inyet another embodiment, the cartridge has a volume of about 1 μl toabout 50 μl. In yet another embodiment, the cartridge has a volume ofabout 5 μl. In yet another embodiment, the cartridge is marked toindicate the composition of the bio-ink. In yet another embodiment, thecartridge is marked to indicate the composition of the support material.In some embodiments, the bio-ink and/or support material is primed. Infurther embodiments, the bio-ink is primed by extruding the bio-ink tothe level of the dispensing orifice prior to initiating the bioprinterprotocol. In further embodiments, the support material is primed byextruding the support material to the level of the dispensing orificeprior to initiating the bioprinter protocol.

Further described herein are systems for attaching a cartridge to abioprinter, comprising: a means for receiving and securing a cartridgeto a printer head of a bioprinter; wherein use of the means forreceiving and securing the cartridge do not substantially alter theposition of the printer head. In some embodiments, the means forreceiving and securing the cartridge to a printer head is selected from:magnetic attraction, a collet chuck grip, a ferrule, a nut, a barreladaptor, or a combination thereof. In one embodiment, in the means forreceiving and securing the cartridge to a printer head is a collet.

Further described herein are receiving surfaces for receiving one ormore structures dispensed from bioprinters. In one embodiment, thereceiving surface is flat or substantially flat. In another embodiment,the receiving surface is smooth or substantially smooth. In yet anotherembodiment, the receiving surface is (a) flat or substantially flat and(b) smooth or substantially smooth or (c) defined or substantiallydefined. In another embodiment, the topography of the receiving surfaceis designed to accommodate or influence the size, shape, or texture, orgeometry one or more dispensed structures. In yet another embodiment,the receiving surface comprises a solid material, a semi-solid material,or a combination thereof. In yet another embodiment, the receivingsurface comprises glass, plastic, metal, a metal alloy, or a combinationthereof. In yet another embodiment, the receiving surface comprises agel. In yet another embodiment, the receiving surface resists adhesionof the one or more structures. In yet another embodiment, the receivingsurface comprises polymerized NovoGel™.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 illustrates a non-limiting example of calibration of a cartridgeusing a horizontal laser.

FIG. 2 illustrates a non-limiting example of calibration of a cartridgeusing a vertical laser.

FIG. 3 illustrates a non-limiting example of a capillary primingprocess.

FIG. 4 illustrates a non-limiting example of a two-dimensionalrepresentation of a bioprinted tissue construct.

FIG. 5 illustrates a non-limiting example of a three-dimensionalconstruct generated by continuous deposition of PF-127 using a NovoGenMMX™ bioprinter connected to a syringe with a 510 μm needle; in thiscase, a pyramid-shaped construct.

FIG. 6 illustrates a non-limiting example of a three-dimensionalconstruct generated by continuous deposition of PF-127 using a NovoGenMMX™ bioprinter connected to a syringe with a 510 μm needle; in thiscase, cube-shaped (left) and hollow cube-shaped (right) constructs.

FIG. 7 illustrates a non-limiting example of a three-dimensionalbioprinter; in this case, a bioprinter including a programmable controlprocessor, a printer head comprising a cartridge, and a means forapplying a wetting agent.

DETAILED DESCRIPTION OF INVENTION

The invention relates to the fields of regenerative medicine,tissue/organ engineering, biologic and medical research, and drugdiscovery. More particularly, the invention relates to devices forfabricating tissues and organs, systems and methods for calibrating andusing such devices, and tissues and organs fabricated by the devices,systems, and methods disclosed herein.

Disclosed herein, in certain embodiments, are bioprinters comprising:one or more printer heads, wherein a printer head comprises a means forreceiving and holding at least one cartridge, and wherein said cartridgecomprises contents selected from one or more of: bio-ink and supportmaterial; a means for calibrating the position of at least onecartridge; and a means for dispensing the contents of at least onecartridge.

Also disclosed herein, in certain embodiments, are methods ofcalibrating the position of a cartridge comprising a deposition orifice,wherein the cartridge is attached to a bioprinter, comprising:calibrating the position of the cartridge along at least one axis;wherein the axis is selected from the x-axis, y-axis, and z-axis; andwherein each calibration is made by use of a laser.

Also disclosed herein, in certain embodiments, are systems forcalibrating the position of a cartridge comprising a deposition orifice,wherein the cartridge is attached to a bioprinter, said systemcomprising: a means for calibrating the position of the cartridge alongat least one axis, wherein the axis is selected from the y-axis, x-axis,and z-axis.

Also disclosed herein, in certain embodiments, are methods forfabricating tissue constructs, comprising: a computer module receivinginput of a visual representation of a desired tissue construct; acomputer module generating a series of commands, wherein the commandsare based on the visual representation and are readable by a bioprinter;a computer module providing the series of commands to a bioprinter; andthe bioprinter depositing bio-ink and support material according to thecommands to form a construct with a defined geometry.

Also disclosed herein, in certain embodiments, are methods of attachinga cartridge to a bioprinter, comprising: (a) inserting the cartridgeinto a collet chuck, wherein the collet chuck is attached to a printerhead of the bioprinter; and (b) adjusting the outer collar of the colletchuck; wherein the inserting and adjusting do not substantially alterthe position of the printer head.

Also disclosed herein, in certain embodiments, are systems for attachinga cartridge to a bioprinter, comprising: a means for receiving andsecuring a cartridge to a printer head of a bioprinter; wherein use ofthe means for receiving and securing the cartridge do not substantiallyalter the position of the printer head.

CERTAIN DEFINITIONS

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural references unless the contextclearly dictates otherwise. Thus, for example, references to “a nucleicacid” includes one or more nucleic acids, and/or compositions of thetype described herein which will become apparent to those personsskilled in the art upon reading this disclosure and so forth. Anyreference to “or” herein is intended to encompass “and/or” unlessotherwise stated.

As used herein, “allograft” means an organ or tissue derived from agenetically non-identical member of the same species as the recipient.

As used herein, “bio-ink” means a liquid, semi-solid, or solidcomposition comprising a plurality of cells. In some embodiments,bio-ink comprises cell solutions, cell aggregates, cell-comprising gels,multicellular bodies, or tissues. In some embodiments, the bio-inkadditionally comprises support material. In some embodiments, thebio-ink additionally comprises non-cellular materials that providespecific biomechanical properties that enable bioprinting.

As used herein, “bioprinting” means utilizing three-dimensional, precisedeposition of cells (e.g., cell solutions, cell-containing gels, cellsuspensions, cell concentrations, multicellular aggregates,multicellular bodies, etc.) via methodology that is compatible with anautomated, computer-aided, three-dimensional prototyping device (e.g., abioprinter).

As used herein, “cartridge” means any object that is capable ofreceiving (and holding) a bio-ink or a support material.

As used herein, a “computer module” means a software component(including a section of code) that interacts with a larger computersystem. In some embodiments, a software module (or program module) comesin the form of a file and typically handles a specific task within alarger software system. In some embodiments, a module may be included inone or more software systems. In other embodiments, a module may beseamlessly integrated with one or more other modules into one or moresoftware systems. A computer module is optionally a stand-alone sectionof code or, optionally, code that is not separately identifiable. A keyfeature of a computer module is that it allows an end user to use acomputer to perform the identified functions.

As used herein, “implantable” means biocompatible and capable of beinginserted or grafted into or affixed onto a living organism eithertemporarily or substantially permanently.

As used herein, “organ” means a collection of tissues joined intostructural unit to serve a common function. Examples of organs include,but are not limited to, skin, sweat glands, sebaceous glands, mammaryglands, bone, brain, hypothalamus, pituitary gland, pineal body, heart,blood vessels, larynx, trachea, bronchus, lung, lymphatic vessel,salivary glands, mucous glands, esophagus, stomach, gallbladder, liver,pancreas, small intestine, large intestine, colon, urethra, kidney,adrenal gland, conduit, ureter, bladder, fallopian tube, uterus,ovaries, testes, prostate, thyroid, parathyroid, meibomian gland,parotid gland, tonsil, adenoid, thymus, and spleen.

As used herein, “patient” means any individual. The term isinterchangeable with “subject,” “recipient,” and “donor.” None of theterms should be construed as requiring the supervision (constant orotherwise) of a medical professional (e.g., physician, nurse, nursepractitioner, physician's assistant, orderly, hospice worker, socialworker, clinical research associate, etc.) or a scientific researcher.

As used herein, “stem cell” means a cell that exhibits potency andself-renewal. Stem cells include, but are not limited to, totipotentcells, pluripotent cells, multipotent cells, oligopotent cells,unipotent cells, and progenitor cells. Stem cells may be embryonic stemcells, peri-natal stem cells, adult stem cells, amniotic stem cells, andinduced pluripotent stem cells.

As used herein, “tissue” means an aggregate of cells. Examples oftissues include, but are not limited to, connective tissue (e.g.,areolar connective tissue, dense connective tissue, elastic tissue,reticular connective tissue, and adipose tissue), muscle tissue (e.g.,skeletal muscle, smooth muscle and cardiac muscle), genitourinarytissue, gastrointestinal tissue, pulmonary tissue, bone tissue, nervoustissue, and epithelial tissue (e.g., simple epithelium and stratifiedepithelium), endoderm-derived tissue, mesoderm-derived tissue, andectoderm-derived tissue.

As used herein, “xenograft” means an organ or tissue derived from adifferent species as the recipient.

Current Methods of Organ Transplants

Currently, there is no reliable method for de novo organ synthesis.Organs are only derived from living donors (e.g., for kidney and liverdonations), deceased donors (e.g., for lung and heart donations) and, ina few cases, animals (e.g., porcine heart valves). Thus, patientsneeding an organ transplant must wait for a donor organ to becomeavailable. This results in a shortage of available organs. Additionally,reliance on organs harvested from a living organism increases the chanceof transplant rejection.

Transplant Rejections

In certain instances, a patient receiving an organ transplant experiencehyperacute rejection. As used herein, “hyperacute rejection” means acomplement-mediated immune response resulting from the recipient'shaving pre-existing antibodies to the donor organ. Hyperacute rejectionoccurs within minutes and is characterized by blood agglutination. Ifthe transplanted organ is not immediately removed, the patient maybecome septic. Xenografts will produce hyperacute rejection unless therecipient is first administered immunosuppressants. In some embodiments,a tissue or organ fabricated de novo will not comprise any antigens andthus cannot be recognized by any antibodies of the recipient.

In certain instances, a patient receiving an organ transplantexperiences acute rejection. As used herein, “acute rejection” means animmune response that begins about one week after transplantation toabout one year after transplantation. Acute rejection results from thepresence of foreign HLA molecules on the donor organ. In certaininstances, APCs recognize the foreign HLAs and activate helper T cells.In certain instances, helper T cells activate cytotoxic T cells andmacrophages. In certain instances, the presence of cytotoxic T cells andmacrophages results in the death of cells with the foreign HLAs and thusdamage (or death) of the transplanted organ. Acute rejection occurs inabout 60-75% of kidney transplants, and 50-60% of liver transplants. Insome embodiments, a tissue or organ fabricated de novo will not compriseany HLAs and thus will not result in the activation of helper T cells.

In certain instances, a patient receiving an organ transplantexperiences chronic rejection. As used herein, “chronic rejection” meanstransplant rejection resulting from chronic inflammatory and immuneresponses against the transplanted tissue. In some embodiments, a tissueor organ fabricated de novo will not comprise any antigens or foreignHLAs and thus will not induce inflammatory or immune responses.

In certain instances, a patient receiving an organ transplantexperiences chronic allograft vasculopathy (CAV). As used herein,“chronic allograft vasculopathy” means loss of function in transplantedorgans resulting from fibrosis of the internal blood vessels of thetransplanted organ. In certain instances, CAV is the result of long-termimmune responses to a transplanted organ. In some embodiments, a tissueor organ fabricated de novo will not comprise any antigens or foreignHLAs and thus will not result in an immune response.

In order to avoid transplant rejection, organ recipients areadministered immunosuppressant drugs. Immunosuppressants include, butare not limited to, corticosteroids (e.g., prednisone andhydrocortisone), calcineurin inhibitors (e.g., cyclosporine andtacrolimus), anti-proliferative agents (e.g., azathioprine andmycophenolic acid), antibodies against specific components of the immunesystem (e.g., basiliximab, dacluzimab, anti-thymocyte globulin (ATG) andanti-lymphocyte globulin (ALG) and mTOR inhibitors (e.g., sirolimus andeverolimus)). However, immunosuppressants have several negativeside-effects including, but not limited to, susceptibility to infection(e.g., infection by pneumocystis carinii pneumonia (PCP),cytomegalovirus pneumonia (CMV), herpes simplex virus, and herpes zostervirus) and the spread of malignant cells, hypertension, dyslipidaemia,hyperglycemia, peptic ulcers, liver and kidney injury, and interactionswith other medicines. In some embodiments, a tissue or organ fabricatedde novo will not result in an immune response and thus will not requirethe administration of an immunosuppressant.

Infections

In certain instances, a donor organ may be infected with an infectiousagent. Following the transplant of the infected organ, the infectiousagent is able to spread throughout the donor (due in part to the use ofimmunosuppressant drugs). By way of non-limiting example, recipientshave contracted HIV, West Nile Virus, rabies, hepatitis C, lymphocyticchoriomeningitis virus (LCMV), tuberculosis, Chagas disease, andCreutzfeldt-Jakob disease from transplanted organs. While suchinfections are rare, they can nevertheless occur—social histories fordeceased donors are often inaccurate as they are necessarily derivedfrom next-of-kin, serological tests may produce false-negative resultsif seroconversion has not occurred, or serological tests may alsoproduce false-negatives due to hemodilution following blood transfusion.Further, many uncommon infectious agents are not screened for due to thelimited time a harvested organ is viable. In some embodiments, a tissueor organ fabricated de novo will not comprise any infectious agents.

Donor Complications

A living donor may also experience complications as a result of donatingan organ. These complications include nosocomial infections, allergicreactions to the anesthesia, and surgical errors. Further, an organdonor may one day find themselves in need of the organ they donated. Forexample, the remaining kidney of a kidney donor or the remaining lobe ofa liver donor may become damaged. In some embodiments, a tissue or organfabricated de novo obviates the need for donor organs and thus willavoid negative side-effects to the donor.

In light of the shortage of available organs and all the complicationsthat can follow a donor organ transplant, there is a need for a methodof de novo fabrication of tissues and organs.

Tissue Engineering

Tissue engineering is an interdisciplinary field that applies andcombines the principles of engineering and life sciences toward thedevelopment of biological substitutes that restore, maintain, or improvetissue function through augmentation, repair, or replacement of an organor tissue. The basic approach to classical tissue engineering is to seedliving cells into a biocompatible and eventually biodegradableenvironment (e.g., a scaffold), and then culture this construct in abioreactor so that the initial cell population can expand further andmature to generate the target tissue upon implantation. With anappropriate scaffold that mimics the biological extracellular matrix(ECM), the developing tissue may adopt both the form and function of thedesired organ after in vitro and in vivo maturation. However, achievinghigh enough cell density with a native tissue-like architecture ischallenging due to the limited ability to control the distribution andspatial arrangement of the cells throughout the scaffold. Theselimitations may result in tissues or organs with poor mechanicalproperties and/or insufficient function. Additional challenges existwith regard to biodegradation of the scaffold, entrapment of residualpolymer, and industrial scale-up of manufacturing processes.Scaffoldless approaches have been attempted. Current scaffoldlessapproaches are subject to several limitations:

-   -   Complex geometries, such as multi-layered structures wherein        each layer comprises a different cell type, may require        definitive, high-resolution placement of cell types within a        specific architecture to reproducibly achieve a native        tissue-like outcome.    -   Scale and geometry are limited by diffusion and/or the        requirement for functional vascular networks for nutrient        supply.    -   The viability of the tissues may be compromised by confinement        material that limits diffusion and restricts the cells' access        to nutrients.

Disclosed herein, in certain embodiments, are devices, systems, andmethods that generate a three-dimensional tissue construct. The devices,systems, and methods disclosed herein utilize a three-phase process: (i)pre-processing, or bio-ink preparation, (ii) processing, i.e. the actualautomated delivery/printing of the bio-ink particles into the bio-paperby the bioprinter, and (iii) post-processing, i.e., thematuration/incubation of the printed construct in the bioreactor. Finalstructure formation takes place during post-processing via the fusion ofthe bio-ink particles. The devices, systems, and methods disclosedherein have the following advantages:

-   -   They are capable of producing cell-comprising tissues and/or        organs.    -   They mimic the environmental conditions of the natural        tissue-forming processes by exploiting principles of        developmental biology.    -   They can achieve a broad array of complex topologies (e.g.,        multilayered structures, repeating geometrical patterns,        segments, sheets, tubes, sacs, etc.).    -   They are compatible with automated means of manufacturing and        are scalable.

Bioprinting enables improved methods of generating cell-comprisingimplantable tissues that are useful in tissue repair, tissueaugmentation, tissue replacement, and organ replacement. Additionally,bioprinting enables improved methods of generating micro-scale tissueanalogs including those useful for in vitro assays.

Bioprinting

Disclosed herein, in certain embodiments, are devices, systems, andmethods for fabricating tissues and organs. In some embodiments, thedevices are bioprinters. In some embodiments, the methods comprise theuse bioprinting techniques. In further embodiments, the tissues andorgans fabricated by use of the devices, systems, and methods describedherein are bioprinted.

In some embodiments, bioprinted cellular constructs, tissues, and organsare made with a method that utilizes a rapid prototyping technologybased on three-dimensional, automated, computer-aided deposition ofcells, including cell solutions, cell suspensions, cell-comprising gelsor pastes, cell concentrations, multicellular bodies (e.g., cylinders,spheroids, ribbons, etc.), and support material onto a biocompatiblesurface (e.g., composed of hydrogel and/or a porous membrane) by athree-dimensional delivery device (e.g., a bioprinter). As used herein,in some embodiments, the term “engineered,” when used to refer totissues and/or organs means that cells, cell solutions, cellsuspensions, cell-comprising gels or pastes, cell concentrates,multicellular aggregates, and layers thereof are positioned to formthree-dimensional structures by a computer-aided device (e.g., abioprinter) according to computer code. In further embodiments, thecomputer script is, for example, one or more computer programs, computerapplications, or computer modules. In still further embodiments,three-dimensional tissue structures form through the post-printingfusion of cells or multicellular bodies similar to self-assemblyphenomena in early morphogenesis.

While a number of methods are available to arrange cells, multicellularaggregates, and/or layers thereof on a biocompatible surface to producea three-dimensional structure including manual placement, positioning byan automated, computer-aided machine such as a bioprinter isadvantageous. Advantages of delivery of cells or multicellular bodieswith this technology include rapid, accurate, and reproducible placementof cells or multicellular bodies to produce constructs exhibitingplanned or pre-determined orientations or patterns of cells,multicellular aggregates and/or layers thereof with variouscompositions. Advantages also include assured high cell density, whileminimizing cell damage.

In some embodiments, methods of bioprinting are continuous and/orsubstantially continuous. A non-limiting example of a continuousbioprinting method is to dispense bio-ink from a bioprinter via adispense tip (e.g., a syringe, capillary tube, etc.) connected to areservoir of bio-ink. In further non-limiting embodiments, a continuousbioprinting method is to dispense bio-ink in a repeating pattern offunctional units. In various embodiments, a repeating functional unithas any suitable geometry, including, for example, circles, squares,rectangles, triangles, polygons, and irregular geometries. In furtherembodiments, a repeating pattern of bioprinted function units comprisesa layer and a plurality of layers are bioprinted adjacently (e.g.,stacked) to form an engineered tissue or organ. In various embodiments,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more layers arebioprinted adjacently (e.g., stacked) to form an engineered tissue ororgan.

In some embodiments, a bioprinted functional unit repeats in atessellated pattern. A “tessellated pattern” is a plane of figures thatfills the plane with no overlaps and no gaps. An advantage of continuousand/or tessellated bioprinting can include an increased production ofbioprinted tissue. Increased production can include achieving increasedscale, increased complexity, or reduced time or cost of production.Another non-limiting potential advantage can be reducing the number ofcalibration events that are required to complete the bioprinting of athree-dimensional construct. Continuous bioprinting may also facilitateprinting larger tissues from a large reservoir of bio-ink, optionallyusing a syringe mechanism.

Methods in continuous bioprinting may involve optimizing and/orbalancing parameters such as print height, pump speed, robot speed, orcombinations thereof independently or relative to each other. In oneexample, the bioprinter head speed for deposition was 3 mm/s, with adispense height of 0.5 mm for the first layer and dispense height wasincreased 0.4 mm for each subsequent layer. In some embodiments, thedispense height is approximately equal to the diameter of the bioprinterdispense tip. Without limitation a suitable and/or optimal dispensedistance does not result in material flattening or adhering to thedispensing needle. In various embodiments, the bioprinter dispense tiphas an inner diameter of about, 20, 50, 100, 150, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 μm, ormore, including increments therein. In various embodiments, the bio-inkreservoir of the bioprinter has a volume of about 0.5, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100 cubic centimeters, or more, including increments therein.The pump speed may be suitable and/or optimal when the residual pressurebuild-up in the system is low. Favorable pump speeds may depend on theratio between the cross-sectional areas of the reservoir and dispenseneedle with larger ratios requiring lower pump speeds. In someembodiments, a suitable and/or optimal print speed enables thedeposition of a uniform line without affecting the mechanical integrityof the material.

The inventions disclosed herein include business methods. In someembodiments, the speed and scalability of the devices and methodsdisclosed herein are utilized to design, build, and operate industrialand/or commercial facilities for production of engineered tissues and/ororgans. In further embodiments, the engineered tissues and/or organs areproduced, stored, distributed, marketed, advertised, and sold as, forexample, materials, tools, and kits for medical treatment of tissuedamage, tissue disease, and/or organ failure or materials, tools, andkits to conduct biological assays and/or drug screening as a service.

Bioprinter

Disclosed herein, in certain embodiments, are bioprinters forfabricating tissues and organs. In some embodiments, a bioprinter is anyinstrument that automates a bioprinting process. In certain embodiments,a bioprinter disclosed herein comprises: one or more printer heads,wherein a printer head comprises a means for receiving and holding atleast one cartridge, and wherein said cartridge comprises contentsselected from one or more of: bio-ink and support material; a means forcalibrating the position of at least one cartridge; and a means fordispensing the contents of at least one cartridge.

In various embodiments, a bioprinter dispenses bio-ink and/or supportmaterial in pre-determined geometries (e.g., positions, patterns, etc.)in two or three dimensions. In some embodiments, a bioprinter achieves aparticular geometry by moving a printer head relative to a printer stageor receiving surface adapted to receive bioprinted materials. In otherembodiments, a bioprinter achieves a particular geometry by moving aprinter stage or receiving surface relative to a printer head. Incertain embodiments, the bioprinter is maintained in a sterileenvironment.

In some embodiments, a bioprinter disclosed herein comprises one or moreprinter heads. In further embodiments, a printer head comprises a meansfor receiving and holding at least one cartridge. In some embodiments, aprinter head comprises a means for receiving and holding more than onecartridge. In some embodiments, the means for receiving and holding atleast one cartridge is selected from: magnetic attraction, a colletchuck grip, a ferrule, a nut, a barrel adapter, or a combinationthereof. In some embodiments, the means for receiving and holding atleast one cartridge is a collet chuck grip.

In some embodiments, a bioprinter disclosed herein comprises a means forcalibrating the position of at least one cartridge. In some embodiments,the means for calibrating the position of at least one cartridge of isselected from: laser alignment, optical alignment, mechanical alignment,piezoelectric alignment, magnetic alignment, electrical field orcapacitance alignment, ultrasound alignment, or a combination thereof.In some embodiments, the means for calibrating the position of at leastone cartridge is laser alignment.

In some embodiments, a bioprinter disclosed herein comprises a means fordispensing the contents of at least one cartridge. In some embodiments,the means for dispensing the contents of at least one cartridge isapplication of a piston, application of pressure, application ofcompressed gas, application of hydraulics, or application of acombination thereof. In some embodiments, the means for dispensing thecontents of at least one cartridge is application of a piston. In someembodiments, the diameter of the piston is less than the diameter of acartridge.

In some embodiments, a bioprinter disclosed herein further comprises areceiving surface. In further embodiments, a receiving surface is anon-cytotoxic surface onto which a bioprinter dispenses bio-ink and/orsupport material. In some embodiments, a bioprinter disclosed hereinfurther comprises a printer stage. In further embodiments, a receivingsurface is a surface of a printer stage. In other embodiments, areceiving surface is component separate from a printer stage, but isaffixed to or supported by a stage. In some embodiments the receivingsurface is flat or substantially flat. In some embodiments the surfaceis smooth or substantially smooth. In other embodiments, the surface isboth substantially flat and substantially smooth. In still furtherembodiments the receiving surface is designed specifically toaccommodate the shape, size, texture, or geometry of the bioprintedstructure. In still further embodiments, the receiving surface controlsor influences the size, shape, texture, or geometry of a bioprintedconstruct.

In some embodiments, a bioprinter disclosed herein further comprises ameans for adjusting temperature. In some embodiments, the means foradjusting temperature adjusts and/or maintains the ambient temperature.In other various embodiments, the means for adjusting temperatureadjusts and/or maintains the temperature of, by way of non-limitingexample, the print head, cartridge, contents of the cartridge (e.g.,bio-ink, support material, etc.), the printer stage, and the receivingsurface.

In some embodiments, the means for adjusting temperature is a heatingelement. In some embodiments, the means for adjusting temperature is aheater. In some embodiments, the means for adjusting temperature is aradiant heater, a convection heater, a conductive heater, a fan heater,a heat exchanger, or a combination thereof. In some embodiments, themeans for adjusting temperature is a cooling element. In someembodiments, the means for adjusting temperature is a container ofcoolant, a chilled liquid, ice, or a combination thereof. In someembodiments, the means for adjusting temperature is a radiant cooler,convection cooler, a conductive cooler, a fan cooler, or a combinationthereof.

In various embodiments, the means for adjusting temperature adjusts atemperature to about 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, or 90° C. including increments therein. In someembodiments, temperature is adjusted to between about 40° C. and about90° C. In other embodiments, temperature is adjusted to between about 0°C. and about 10° C.

In some embodiments, a bioprinter disclosed herein, further comprises ameans for applying a wetting agent to one or more of: the printer stage;the receiving surface, the deposition orifice, bio-ink, supportmaterial, or the printed construct. In some embodiments, the means forapplying the wetting agent is any suitable method of applying a fluid(e.g., a sprayer, a pipette, an inkjet, etc.). In some embodiments, thewetting agent is water, tissue culture media, buffered salt solutions,serum, or a combination thereof. In further embodiments, a wetting agentis applied after the bio-ink or supporting material is dispensed by thebioprinter. In some embodiments, a wetting agent is appliedsimultaneously or substantially simultaneously with the bio-ink orsupporting material being dispensed by the bioprinter. In someembodiments, a wetting agent is applied prior to the bio-ink orsupporting material being dispensed by the bioprinter.

Printer Head

Disclosed herein, in certain embodiments, are bioprinters forfabricating tissues and organs. In some embodiments, a bioprinterdisclosed herein comprises one or more printer heads. In furtherembodiments, a printer head comprises a means for receiving and holdingat least one cartridge. In still further embodiments, a printer headattaches at least one cartridge to a bioprinter.

Many means for receiving and holding at least one cartridge aresuitable. Suitable means for receiving and holding at least onecartridge include those that reliably, precisely, and securely attach atleast one cartridge to a bioprinter. In various embodiments, the meansfor receiving and holding at least one cartridge is, by way ofnon-limiting example, magnetic attraction, a collet chuck grip, aferrule, a nut, a barrel adapter, or a combination thereof.

In some embodiments, a printer head disclosed herein receives and holdsone cartridge. In various other embodiments, a printer head disclosedherein receives and holds 2, 3, 4, 5, 6, 7, 8, 9, 10, or more cartridgessimultaneously. In further embodiments, a printer head disclosed hereinfurther comprises a means to select a cartridge to be employed inbioprinting from among a plurality of cartridges received and held.

In some embodiments, a printer head disclosed herein further comprises(or is in fluid communication with) a reservoir to contain bio-inkand/or support materials beyond the capacity of the one or morecartridges. In further embodiments, a reservoir supplies bio-ink and/orsupport materials to one or more cartridges for dispensing via adispensing orifice. Printer head configurations including a reservoirare particularly useful in continuous or substantially continuousbioprinting applications. Many volumes are suitable for a reservoirdisclosed herein. In various embodiments, a reservoir has an internalvolume of, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250,300, 350, 400, 450, 500 ml or more, including increments therein.

In some embodiments, bioprinting involves using a computer to configureparameters such as print height, pump speed, robot speed, orcombinations thereof independently or relative to each other. In furtherembodiments, computer code specifies the positioning of a printer headto configure printer head height above a receiving surface. In furtherembodiments, computer code specifies the direction and speed of themotion of a printer head to configure dispensing characteristics forbio-ink and/or support material.

Cartridges

Disclosed herein, in certain embodiments, are bioprinters forfabricating tissues and organs. In some embodiments, a cartridgeattached to the bioprinter comprises bio-ink or support material. Insome embodiments, the bioprinter dispenses bio-ink or support materialin a specific pattern and at specific positions in order to form aspecific cellular construct, tissue, or organ. In order to fabricatecomplex tissue constructs, the bioprinter deposits the bio-ink orsupport material at precise speeds and in uniform amounts. Thus, thereis a need for a cartridge with (a) a dispensing orifice that is smoothor substantially smooth, and (b) an internal surface that is smooth orsubstantially smooth. As used herein, “cartridge” means any object thatis capable of receiving (and holding) a bio-ink and/or support material.

In some embodiments, a cartridge disclosed herein comprises bio-ink. Insome embodiments, a cartridge disclosed herein comprises supportmaterial. In some embodiments, a cartridge disclosed herein comprises acombination of bio-ink and support material.

Disclosed herein, in certain embodiments, are cartridges for use with abioprinter disclosed herein, comprising at least one dispensing orifice.In some embodiments, a cartridge comprises one dispensing orifice. Invarious other embodiments, a cartridge comprises 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or moredispensing orifices. In further embodiment, the edges of a dispensingorifice are smooth or substantially smooth.

Many shapes are suitable for the dispensing orifices disclosed herein.In various embodiments, suitable shapes for dispensing orifices include,by way of non-limiting examples, circular, ovoid, triangular, square,rectangular, polygonal, and irregular. In some embodiments, the orificeis circular. In other embodiments, the orifice is square. In yet otherembodiments, the orifice is oval, oblong, or rectangular and dispensessolid or semi-solid bio-ink and/or support materials in a ribbon-likeform.

In some embodiments, the internal surface of the cartridge is smooth orsubstantially smooth. In some embodiments, the cartridge is comprised ofa rigid structure to facilitate calibration. In some embodiments, thewalls of the cartridge are comprised of a material that resistsattachment of cells. In some embodiments, the cartridges are comprisedof a material that is biocompatible.

Many types of cartridges are suitable for use with bioprinters disclosedherein and the methods of using the same. In some embodiments, acartridge is compatible with bioprinting that involves extruding asemi-solid or solid bio-ink or a support material through one or moredispensing orifices. In some embodiments, a cartridge is compatible withbioprinting that involves dispensing a liquid or semi-solid cellsolution, cell suspension, or cell concentration through one or moredispensing orifices. In some embodiments, a cartridge is compatible withnon-continuous bioprinting. In some embodiments, a cartridge iscompatible with continuous and/or substantially continuous bioprinting.

In some embodiments, a cartridge is a capillary tube or a micropipette.In some embodiments, a cartridge is a syringe or a needle. Many internaldiameters are suitable for substantially round or cylindricalcartridges. In various embodiments, suitable internal diameters include,by way of non-limiting examples, 1, 10, 50, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000 or more μm, including increments therein. Inother various embodiments, suitable internal diameters include, by wayof non-limiting examples, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50,60, 70, 80, 90, 100 or more mm, including increments therein. In someembodiments, a cartridge has an internal diameter of about 1 μm to about1000 μm. In a particular embodiment, a cartridge has an internaldiameter of about 500 μm. In another particular embodiment, a cartridgehas an internal diameter of about 250 μm. Many internal volumes aresuitable for the cartridges disclosed herein. In various embodiments,suitable internal volumes include, by way of non-limiting examples, 1,10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 ormore μl, including increments therein. In other various embodiments,suitable internal volumes include, by way of non-limiting examples, 1,2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500or more ml, including increments therein. In some embodiments, acartridge has a volume of about 1 μl to about 50 μl. In a particularembodiment, a cartridge has a volume of about 5 μl.

In some embodiments, a cartridge is compatible with ink jet printing ofbio-ink and/or support material onto a receiving surface such as thatdescribed in U.S. Pat. No. 7,051,654. In further embodiments, acartridge includes dispensing orifices in the form of voltage-gatednozzles or needles under the control of the computer code describedherein.

In some embodiments, the cartridge is primed. In some embodiments,priming the cartridge increases the accuracy of the dispensing,deposition, or extrusion process. As used herein, “primed” means thecontents of the cartridge are made ready for dispensing, deposition, orextrusion by compacting and advancing the contents of the cartridgeuntil the material to be dispensed (bio-ink or supporting material) islocated in a position in contact with the dispensing orifice. In someembodiments, the cartridge is primed when the contents are compact orsubstantially compact, and the contents are in physical contact with theorifice of the cartridge.

In some embodiments, a cartridge is marked to indicate the compositionof its contents. In further embodiments, a cartridge is marked toindicate the composition of a bio-ink and/or support material containedtherein. In some embodiments, the surface of the cartridge is colored.In some embodiments, the outer surface of the cartridge is dyed,painted, marked with a pen, marked by a sticker, or a combinationthereof.

In some embodiments, the outer surface of a cartridge is marked toincrease the opacity of the surface of the cartridge (e.g., to increasethe amount of a laser beam that is reflected off the surface of thecartridge). In some embodiments, the surface of a cartridge is colored.In some embodiments, the outer surface of a cartridge is scored. As usedherein, “scored” means marking the surface of a cartridge to reduce thesmoothness of the surface. Any suitable method is used to score thesurface of a cartridge (e.g., application of an acidic substance,application of a caustic substance, application of an abrasivesubstance, etc.). In some embodiments, the outer surface of a cartridgeis painted, polished (e.g., fire polished), etched (e.g., laser etched),marked with a pen, marked by a sticker, or a combination thereof.

Grip

Disclosed herein, in certain embodiments, are bioprinters forfabricating tissues and organs. In some embodiments, a cartridgeattached to a bioprinter comprises bio-ink and/or support material. Insome embodiments, the bioprinter dispenses the bio-ink and/or supportmaterial in a specific pattern and at specific positions in order toform a specific cellular construct, tissue, or organ. In someembodiments, a cartridge comprising bio-ink is disposable. In someembodiments, the cartridge is ejected from the bioprinter followingextrusion of the contents. In some embodiments, a new cartridge issubsequently attached to the bioprinter.

In order to fabricate complex structures, the bioprinters disclosedherein dispense bio-ink and/or support material from a cartridge with asuitable repeatable accuracy. In various embodiments, suitablerepeatable accuracies include those of about ±5, 10, 20, 30, 40, or 50μm on any axis. In some embodiments, the bioprinters disclosed hereindispense bio-ink and/or support material from a cartridge with arepeatable accuracy of about ±20 μm. However, uncontrolled removal andinsertion of cartridges can result in alterations of the position of theprinter head (and thus the cartridges) with respect to the tissueconstruct, such that precision of the placement of the first bio-inkparticle deposited from a new cartridge may vary relative to the lastbio-ink particle deposited from the previous cartridge. Thus, there is aneed for a method of attaching and securing a cartridge to a printerhead, wherein said attaching and securing produce minimal alterations inthe position of the printer head.

Disclosed herein, in certain embodiments, are methods of attaching acartridge to a bioprinter, comprising: (a) inserting the cartridge intoa collet chuck, wherein the collet chuck is attached to a printer headof the bioprinter; and (b) adjusting the outer collar of the colletchuck; wherein the inserting and adjusting do not substantially alterthe position of the printer head.

Disclosed herein, in certain embodiments, are systems for attaching acartridge to a bioprinter, comprising: a means for receiving andsecuring a cartridge to a printer head of a bioprinter; wherein use ofthe means for receiving and securing the cartridge do not substantiallyalter the position of the printer head. In some embodiments, the meansfor receiving and securing the cartridge to a printer head is a chuck orferrule. As used herein, “chuck” means a holding device consisting ofadjustable jaws. In some embodiments, the means for receiving andsecuring the cartridge to a printer head is a collet. As used herein,“collet” means a subtype of chuck—that forms a collar around the objectto be held and exerts a strong clamping. As used herein, “ferrule” meansa band (e.g., a metal band) used to secure one object to another. Insome embodiments, the means for receiving and securing the cartridge toa printer head is a barrel adaptor. As used herein, “barrel adaptor”means a threaded tube used to secure one object to another.

Receiving Surface

Disclosed herein, in certain embodiments, are bioprinters forfabricating tissues and organs. In some embodiments, the bioprinterdispenses a plurality of elements, sections, and/or areas of bio-inkand/or support material onto a receiving surface. In furtherembodiments, dispensing occurs in a specific pattern and at specificpositions. In still further embodiments, the locations at which thebioprinter deposits bio-ink and/or support material onto a receivingsurface are defined by user input and translated into computer code.

In some embodiments, each of the elements, sections, and/or areas ofbio-ink and/or support material has dimensions of less than 300 mm×300mm×160 mm. By way of example only, the dimensions of a section ofbio-ink or support material may be 75 mm×5.0 mm×5.0 mm; 0.3 mm×2.5mm×2.5 mm; 1 mm×1 mm×50 mm; or 150 mm×150 mm×80 mm. Due to the generallysmall size of each section, and in some cases, the high degree ofprecision required, minute imperfections in the receiving surface mayresult in imperfections (and possibly, failure) of a cellular construct,tissue, or organ. Thus, there is a need for a substantially smooth andsubstantially flat receiving surface, or a defined or substantiallydefined receiving surface, that is able to receive sections of bio-inkand/or support material.

Disclosed herein, in certain embodiments, are receiving surfaces forreceiving one or more structures generated by the bioprinter disclosedherein. In some embodiments, the receiving surface is flat orsubstantially flat. In some embodiments, the receiving surface is smoothor substantially smooth. In some embodiments, the receiving surface isflat or substantially flat. In some embodiments, the receiving surfaceis defined or substantially defined. In other embodiments the receivingsurface is designed specifically to accommodate the shape, size,texture, or geometry of a specific bioprinted structure. In furtherembodiments, the receiving surface controls or influences the size,shape, texture, or geometry of a bioprinted construct.

In some embodiments, the receiving surface comprises a solid material, asemi-solid material, or a combination thereof. In some embodiments, thereceiving surface comprises glass, coated glass, plastic, coatedplastic, metal, a metal alloy, or a combination thereof. In someembodiments, the receiving surface comprises a gel. In some embodiments,the receiving surface and any coatings thereon are biocompatible. Invarious embodiments, the receiving surface comprises any of the supportmaterials and/or confinement materials disclosed herein. In specificembodiments, the receiving surface comprises polymerized NovoGel™ orpolymerized agarose, polymerized gelatin, extracellular matrix (orcomponents thereof), collagen, or a combination thereof.

Software

Disclosed herein, in certain embodiments, are bioprinters forfabricating tissues and organs. In some embodiments, one or morecartridges attached to the bioprinter comprises bio-ink and/or supportmaterial. In some embodiments, the bioprinter dispenses bio-ink orsupport material in a specific pattern and at specific positions inorder to form a specific cellular construct, tissue, or organ.

In order to fabricate complex tissue constructs, the bioprinter depositsthe bio-ink or support material at precise locations (in two or threedimensions) on a receiving surface. In some embodiments, the locationsat which the bioprinter deposits bio-ink and/or support material aredefined by user input and translated into computer code. In furtherembodiments, the computer code includes a sequence of instructions,executable in the central processing unit (CPU) of a digital processingdevice, written to perform a specified task. In some embodiments,additional bioprinting parameters including, by way of non-limitingexamples, print height, pump speed, robot speed, and/or control ofvariable dispensing orifices are defined by user input and translatedinto computer code. In other embodiments, such bioprinting parametersare not directly defined by user input, but are derived from otherparameters and conditions by the computer code described herein.

Disclosed herein, in certain embodiments, are methods for fabricatingtissue constructs, tissues, and organs, comprising: a computer modulereceiving input of a visual representation of a desired tissueconstruct; a computer module generating a series of commands, whereinthe commands are based on the visual representation and are readable bya bioprinter; a computer module providing the series of commands to abioprinter; and the bioprinter depositing bio-ink and/or supportmaterial according to the commands to form a construct with a definedgeometry.

Computer Readable Medium

In some embodiments, the locations at which the bioprinter deposits thebio-ink and/or support material are defined by user input and translatedinto computer code. In some embodiments, the devices, systems, andmethods disclosed herein further comprise computer readable media ormedia encoded with computer readable program code. In furtherembodiments, a computer readable medium is a tangible component of adigital processing device such as a bioprinter (or a component thereof)or a computer connected to a bioprinter (or a component thereof). Instill further embodiments, a computer readable medium is optionallyremovable from a digital processing device. In some embodiments, acomputer readable medium includes, by way of non-limiting examples,CD-ROMs, DVDs, flash memory devices, solid state memory, magnetic diskdrives, magnetic tape drives, optical disk drives, cloud computingsystems and services, and the like.

Computer Modules

In some embodiments, the devices, systems, and methods described hereincomprise software, server, and database modules. In some embodiments, a“computer module” is a software component (including a section of code)that interacts with a larger computer system. In further embodiments, asoftware module (or program module) comes in the form of one or morefiles and typically handles a specific task within a larger softwaresystem.

In some embodiments, a module is included in one or more softwaresystems. In other embodiments, a module is integrated with one or moreother modules into one or more software systems. A computer module isoptionally a stand-alone section of code or, optionally, code that isnot separately identifiable. In some embodiments, the modules are in asingle application. In other embodiments, the modules are in a pluralityof applications. In some embodiments, the modules are hosted on onemachine. In other embodiments, the modules are hosted on a plurality ofmachines. In some embodiments, the modules are hosted on a plurality ofmachines in one location. In other embodiments, the modules are hosted aplurality of machines in more than one location. Further describedherein is the formatting of location and positioning data. In someembodiments, the data files described herein are formatted in anysuitable data serialization format including, by way of non-limitingexamples, tab-separated values, comma-separated values,character-separated values, delimiter-separated values, XML, JSON, BSON,and YAML. A key feature of a computer module is that it allows an enduser to use a computer to perform the identified functions.

Graphic User Interface

In some embodiments, a computer module comprises a graphic userinterface (GUI). As used herein, “graphic user interface” means a userenvironment that uses pictorial as well as textual representations ofthe input and output of applications and the hierarchical or other datastructure in which information is stored. In some embodiments, acomputer module comprises a display screen. In further embodiments, acomputer module presents, via a display screen, a two-dimensional GUI.In other embodiments, a computer module presents, via a display screen,a three-dimensional GUI such as a virtual reality environment. In someembodiments, the display screen is a touchscreen or multitouchscreen andpresents an interactive GUI.

In some embodiments, the display screen presents a GUI that consistsessentially of a grid comprising regularly spaced objects ofsubstantially the same shape and substantially equal size. The objectspresented have any suitable shape. In some embodiments, suitable shapesfor objects include, by way of non-limiting examples, circle, oval,square, rectangle, triangle, diamond, polygon, or a combination thereof.

In some embodiments, a user defines the content of one or more objectsto form a two-dimensional or three-dimensional visual representation ofa desired tissue construct. See, e.g., FIG. 4. In some embodiments, theuser-defined content of an object is, by way of non-limiting examples, abio-ink with various compositions or support material with variouscompositions. In some embodiments, the user defines the content of anobject by modifying the color of the cell or the shape of the object.

Bio-Ink

Disclosed herein, in certain embodiments, are devices, systems, andmethods for fabricating tissues and organs. In some embodiments, thedevices comprise one or more printer heads for receiving and holding atleast one cartridge that optionally contains bio-ink. In someembodiments, the methods comprise the use of bio-ink. In furtherembodiments, the tissues and organs fabricated by use of the devices,systems, and methods described herein comprise bio-ink at the time offabrication or thereafter.

In some embodiments, “bio-ink” includes liquid, semi-solid, or solidcompositions comprising a plurality of cells. In some embodiments,bio-ink comprises liquid or semi-solid cell solutions, cell suspensions,or cell concentrations. In further embodiments, a cell solution,suspension, or concentration comprises a liquid or semi-solid (e.g.,viscous) carrier and a plurality of cells. In still further embodiments,the carrier is a suitable cell nutrient media, such as those describedherein. In some embodiments, bio-ink comprises semi-solid or solidmulticellular aggregates or multicellular bodies. In furtherembodiments, the bio-ink is produced by 1) mixing a plurality of cellsor cell aggregates and a biocompatible liquid or gel in a pre-determinedratio to result in bio-ink, and 2) compacting the bio-ink to produce thebio-ink with a desired cell density and viscosity. In some embodiments,the compacting of the bio-ink is achieved by centrifugation, tangentialflow filtration (“TFF”), or a combination thereof. In some embodiments,the compacting of the bio-ink results in a composition that isextrudable, allowing formation of multicellular aggregates ormulticellular bodies. In some embodiments, “extrudable” means able to beshaped by forcing (e.g., under pressure) through a nozzle or orifice(e.g., one or more holes or tubes). In some embodiments, the compactingof the bio-ink results from growing the cells to a suitable density. Thecell density necessary for the bio-ink will vary with the cells beingused and the tissue or organ being produced. In some embodiments, thecells of the bio-ink are cohered and/or adhered. In some embodiments,“cohere,” “cohered,” and “cohesion” refer to cell-cell adhesionproperties that bind cells, multicellular aggregates, multicellularbodies, and/or layers thereof. In further embodiments, the terms areused interchangeably with “fuse,” “fused,” and “fusion.” In someembodiments, the bio-ink additionally comprises support material, cellculture medium, extracellular matrix (or components thereof), celladhesion agents, cell death inhibitors, anti-apoptotic agents,anti-oxidants, extrusion compounds, and combinations thereof.

Cells

Disclosed herein, in various embodiments, are bio-inks that includeliquid, semi-solid, or solid compositions comprising a plurality ofcells. In some embodiments, bio-ink comprises liquid or semi-solid cellsolutions, cell suspensions, or cell concentrations. In someembodiments, any mammalian cell is suitable for use in bio-ink and inthe fabrication of tissues and organs using the devices, systems, andmethods described herein. In various embodiments, the cells are anysuitable cell. In further various embodiments, the cells are vertebratecells, mammalian cells, human cells, or combinations thereof. In someembodiments, the type of cell used in a method disclosed herein dependson the type of cellular construct, tissue, or organ being produced. Insome embodiments, the bio-ink comprises one type of cell (also referredto as a “homologous ink”). In some embodiments, the bio-ink comprisesmore than one type of cell (also referred to as a “heterologous ink”).

In further embodiments, the cells are, by way of non-limiting examples,contractile or muscle cells (e.g., skeletal muscle cells,cardiomyocytes, smooth muscle cells, and myoblasts), connective tissuecells (e.g., bone cells, cartilage cells, fibroblasts, and cellsdifferentiating into bone forming cells, chondrocytes, or lymphtissues), bone marrow cells, endothelial cells, skin cells, epithelialcells, breast cells, vascular cells, blood cells, lymph cells, neuralcells, Schwann cells, gastrointestinal cells, liver cells, pancreaticcells, lung cells, tracheal cells, corneal cells, genitourinary cells,kidney cells, reproductive cells, adipose cells, parenchymal cells,pericytes, mesothelial cells, stromal cells, undifferentiated cells(e.g., embryonic cells, stem cells, and progenitor cells),endoderm-derived cells, mesoderm-derived cells, ectoderm-derived cells,and combinations thereof.

In some embodiments, the cells are adult, differentiated cells. Infurther embodiments, “differentiated cells” are cells with atissue-specific phenotype consistent with, for example, a smooth musclecell, a fibroblast, or an endothelial cell at the time of isolation,wherein tissue-specific phenotype (or the potential to display thephenotype) is maintained from the time of isolation to the time of use.In other embodiments, the cells are adult, non-differentiated cells. Infurther embodiments, “non-differentiated cells” are cells that do nothave, or have lost, the definitive tissue-specific traits of forexample, smooth muscle cells, fibroblasts, or endothelial cells. In someembodiments, non-differentiated cells include stem cells. In furtherembodiments, “stem cells” are cells that exhibit potency andself-renewal. Stem cells include, but are not limited to, totipotentcells, pluripotent cells, multipotent cells, oligopotent cells,unipotent cells, and progenitor cells. Stem cells may be embryonic stemcells, adult stem cells, amniotic stem cells, and induced pluripotentstem cells. In yet other embodiments, the cells are a mixture of adult,differentiated cells and adult, non-differentiated cells.

Cell Culture Media

In some embodiments, the bio-ink comprises a cell culture medium. Thecell culture medium is any suitable medium. In various embodiments,suitable cell culture media include, by way of non-limiting examples,Dulbecco's Phosphate Buffered Saline, Earle's Balanced Salts, Hanks'Balanced Salts, Tyrode's Salts, Alsever's Solution, Gey's Balanced SaltSolution, Kreb's-Henseleit Buffer Modified, Kreb's-Ringer BicarbonateBuffer, Puck's Saline, Dulbecco's Modified Eagle's Medium, Dulbecco'sModified Eagle's Medium/Nutrient F-12 Ham, Nutrient Mixture F-10 Ham(Ham's F-10), Medium 199, Minimum Essential Medium Eagle, RPMI-1640Medium, Ames' Media, BGJb Medium (Fitton-Jackson Modification), Click'sMedium, CMRL-1066 Medium, Fischer's Medium, Glascow Minimum EssentialMedium (GMEM), Iscove's Modified Dulbecco's Medium (IMDM), L-15 Medium(Leibovitz), McCoy's 5A Modified Medium, NCTC Medium, Swim's S-77Medium, Waymouth Medium, William's Medium E, or combinations thereof. Insome embodiments, the cell culture medium is modified or supplemented.In some embodiments, the cell culture medium further comprises albumin,selenium, transferrins, fetuins, sugars, amino acids, vitamins, growthfactors, cytokines, hormones, antibiotics, lipids, lipid carriers,cyclodextrins, or a combination thereof.

Extracellular Matrix

In some embodiments, the bio-ink further comprises one or morecomponents of an extracellular matrix or derivatives thereof. In someembodiments, “extracellular matrix” includes proteins that are producedby cells and transported out of the cells into the extracellular space,where they may serve as a support to hold tissues together, to providetensile strength, and/or to facilitate cell signaling. Examples, ofextracellular matrix components include, but are not limited to,collagen, fibronectin, laminin, hyaluronates, elastin, andproteoglycans. For example, multicellular aggregates may contain variousECM proteins (e.g., gelatin, fibrinogen, fibrin, collagen, fibronectin,laminin, elastin, and/or proteoglycans). The ECM components orderivatives of ECM components can be added to the cell paste used toform the multicellular aggregate. The ECM components or derivatives ofECM components added to the cell paste can be purified from a human oranimal source, or produced by recombinant methods known in the art.Alternatively, the ECM components or derivatives of ECM components canbe naturally secreted by the cells in the elongate cellular body, or thecells used to make the elongate cellular body can be geneticallymanipulated by any suitable method known in the art to vary theexpression level of one or more ECM components or derivatives of ECMcomponents and/or one or more cell adhesion molecules or cell-substrateadhesion molecules (e.g., selectins, integrins, immunoglobulins, andadherins). The ECM components or derivatives of ECM components maypromote cohesion of the cells in the multicellular aggregates. Forexample, gelatin and/or fibrinogen can suitably be added to the cellpaste, which is used to form multicellular aggregates. The fibrinogencan then be converted to fibrin by the addition of thrombin.

In some embodiments, the bio-ink further comprises an agent thatencourages cell adhesion.

In some embodiments, the bio-ink further comprises an agent thatinhibits cell death (e.g., necrosis, apoptosis, or autophagocytosis). Insome embodiments, the bio-ink further comprises an anti-apoptotic agent.Agents that inhibit cell death include, but are not limited to, smallmolecules, antibodies, peptides, peptibodies, or combination thereof. Insome embodiments, the agent that inhibits cell death is selected from:anti-TNF agents, agents that inhibit the activity of an interleukin,agents that inhibit the activity of an interferon, agents that inhibitthe activity of an GCSF (granulocyte colony-stimulating factor), agentsthat inhibit the activity of a macrophage inflammatory protein, agentsthat inhibit the activity of TGF-B (transforming growth factor B),agents that inhibit the activity of an MMP (matrix metalloproteinase),agents that inhibit the activity of a caspase, agents that inhibit theactivity of the MAPK/JNK signaling cascade, agents that inhibit theactivity of a Src kinase, agents that inhibit the activity of a JAK(Janus kinase), or a combination thereof. In some embodiments, thebio-ink comprises an anti-oxidant.

Extrusion Compounds

In some embodiments, the bio-ink further comprises an extrusion compound(i.e., a compound that modifies the extrusion properties of thebio-ink). Examples of extrusion compounds include, but are not limitedto gels, hydrogels, surfactant polyols (e.g., Pluronic F-127 or PF-127),thermo-responsive polymers, hyaluronates, alginates, extracellularmatrix components (and derivatives thereof), collagens, otherbiocompatible natural or synthetic polymers, nanofibers, andself-assembling nanofibers.

Gels, sometimes referred to as jellies, have been defined in variousways. For example, the United States Pharmacopoeia defines gels assemisolid systems consisting of either suspensions made up of smallinorganic particles or large organic molecules interpenetrated by aliquid. Gels include a single-phase or a two-phase system. Asingle-phase gel consists of organic macromolecules distributeduniformly throughout a liquid in such a manner that no apparentboundaries exist between the dispersed macromolecules and the liquid.Some single-phase gels are prepared from synthetic macromolecules (e.g.,carbomer) or from natural gums (e.g., tragacanth). In some embodiments,single-phase gels are generally aqueous, but will also be made usingalcohols and oils. Two-phase gels consist of a network of small discreteparticles.

Gels can also be classified as being hydrophobic or hydrophilic. Incertain embodiments, the base of a hydrophobic gel consists of a liquidparaffin with polyethylene or fatty oils gelled with colloidal silica,or aluminum or zinc soaps. In contrast, the base of hydrophobic gelsusually consists of water, glycerol, or propylene glycol gelled with asuitable gelling agent (e.g., tragacanth, starch, cellulose derivatives,carboxyvinylpolymers, and magnesium-aluminum silicates). In certainembodiments, the rheology of the compositions or devices disclosedherein is pseudo plastic, plastic, thixotropic, or dilatant.

Suitable hydrogels include those derived from collagen, hyaluronate,fibrin, alginate, agarose, chitosan, and combinations thereof. In otherembodiments, suitable hydrogels are synthetic polymers. In furtherembodiments, suitable hydrogels include those derived from poly(acrylicacid) and derivatives thereof, poly(ethylene oxide) and copolymersthereof, poly(vinyl alcohol), polyphosphazene, and combinations thereof.In various specific embodiments, the support material is selected from:hydrogel, NovoGel™, agarose, alginate, gelatin, Matrigel™, hyaluronan,poloxamer, peptide hydrogel, poly(isopropyl n-polyacrylamide),polyethylene glycol diacrylate (PEG-DA), hydroxyethyl methacrylate,polydimethylsiloxane, polyacrylamide, poly(lactic acid), silicon, silk,or combinations thereof.

In some embodiments, hydrogel-based extrusion compounds arethermoreversible gels (also known as thermo-responsive gels orthermogels). In some embodiments, a suitable thermoreversible hydrogelis not a liquid at room temperature. In specific embodiments, thegelation temperature (Tgel) of a suitable hydrogel is about 10° C.,about 15° C., about 20° C., about 25° C., about 30° C., about 35° C.,and about 40° C., including increments therein. In certain embodiments,the Tgel of a suitable hydrogel is about 10° C. to about 25° C. In someembodiments, the bio-ink (e.g., comprising hydrogel, one or more celltypes, and other additives, etc.) described herein is not a liquid atroom temperature. In specific embodiments, the gelation temperature(Tgel) of a bio-ink described herein is about 10° C., about 15° C.,about 20° C., about 25° C., about 30° C., about 35° C., and about 40°C., including increments therein. In certain embodiments, the Tgel of abio-ink described herein is about 10° C. to about 25° C.

Polymers composed of polyoxypropylene and polyoxyethylene formthermoreversible gels when incorporated into aqueous solutions. Thesepolymers have the ability to change from the liquid state to the gelstate at temperatures that can be maintained in a bioprinter apparatus.The liquid state-to-gel state phase transition is dependent on thepolymer concentration and the ingredients in the solution.

Poloxamer 407 (Pluronic F-127 or PF-127) is a nonionic surfactantcomposed of polyoxyethylene-polyoxypropylene copolymers. Otherpoloxamers include 188 (F-68 grade), 237 (F-87 grade), 338 (F-108grade). Aqueous solutions of poloxamers are stable in the presence ofacids, alkalis, and metal ions. PF-127 is a commercially availablepolyoxyethylene-polyoxypropylene triblock copolymer of general formulaE106 P70 E106, with an average molar mass of 13,000. The polymer can befurther purified by suitable methods that will enhance gelationproperties of the polymer. It contains approximately 70% ethylene oxide,which accounts for its hydrophilicity. It is one of the series ofpoloxamer ABA block copolymers. PF-127 has good solubilizing capacity,low toxicity and is, therefore, considered a suitable extrusioncompound.

In some embodiments, the viscosity of the hydrogels and bio-inkspresented herein is measured by any means described. For example, insome embodiments, an LVDV-II+CP Cone Plate Viscometer and a Cone SpindleCPE-40 is used to calculate the viscosity of the hydrogels and bio-inks.In other embodiments, a Brookfield (spindle and cup) viscometer is usedto calculate the viscosity of the hydrogels and bio-inks. In someembodiments, the viscosity ranges referred to herein are measured atroom temperature. In other embodiments, the viscosity ranges referred toherein are measured at body temperature (e.g., at the average bodytemperature of a healthy human).

In further embodiments, the hydrogels and/or bio-inks are characterizedby having a viscosity of between about 500 and 1,000,000 centipoise,between about 750 and 1,000,000 centipoise; between about 1000 and1,000,000 centipoise; between about 1000 and 400,000 centipoise; betweenabout 2000 and 100,000 centipoise; between about 3000 and 50,000centipoise; between about 4000 and 25,000 centipoise; between about 5000and 20,000 centipoise; or between about 6000 and 15,000 centipoise.

In some embodiments, the bio-ink comprises cells and extrusion compoundssuitable for continuous bioprinting. In specific embodiments, thebio-ink has a viscosity of about 1500 mPa·s. A mixture of Pluronic F-127and cellular material may be suitable for continuous bioprinting. Such abio-ink may be prepared by dissolving Pluronic F-127 powder bycontinuous mixing in cold (4° C.) phosphate buffered saline (PBS) over48 hours to 30% (w/v). Pluronic F-127 may also be dissolved in water.Cells may be cultivated and expanded using standard sterile cell culturetechniques. The cells may be pelleted at 200 g for example, andre-suspended in the 30% Pluronic F-127 and aspirated into a reservoiraffixed to a bioprinter where it can be allowed to solidify at agelation temperature from about 10 to about 25° C. Gelation of thebio-ink prior to bioprinting is optional. The bio-ink, including bio-inkcomprising Pluronic F-127 can be dispensed as a liquid.

In various embodiments, the concentration of Pluronic F-127 can be anyvalue with suitable viscosity and/or cytotoxicity properties. A suitableconcentration of Pluronic F-127 may also be able to support weight whileretaining its shape when bioprinted. In some embodiments, theconcentration of Pluronic F-127 is about 10%, about 15%, about 20%,about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%. Insome embodiments, the concentration of Pluronic F-127 is between about30% and about 40%, or between about 30% and about 35%.

FIG. 5 depicts a three-dimensional, pyramid-shaped construct generatedby continuous deposition of PF-127 using a NovoGen MMX™ bioprinterconnected to a syringe with a 510 μm needle.

FIG. 6 depicts a three-dimensional, cube-shaped (left) and hollowcube-shaped (right) constructs generated by continuous deposition ofPF-127 using a NovoGen MMX™ bioprinter connected to a syringe with a 510μm needle.

In some embodiments, the non-cellular components of the bio-ink (e.g.,extrusion compounds, etc.) are removed prior to use. In furtherembodiments, the non-cellular components are, for example, hydrogels,surfactant polyols, thermo-responsive polymers, hyaluronates, alginates,collagens, or other biocompatible natural or synthetic polymers. Instill further embodiments, the non-cellular components are removed byphysical, chemical, or enzymatic means. In some embodiments, aproportion of the non-cellular components remain associated with thecellular components at the time of use.

In some embodiments, the cells are pre-treated to increase cellularinteraction. For example, cells may be incubated inside a centrifugetube after centrifugation in order to enhance cell-cell interactionsprior to shaping the bio-ink.

Support Material

Disclosed herein, in certain embodiments, are devices, systems, andmethods for fabricating tissues and organs. In some embodiments, thedevices comprise one or more printer heads for receiving and holding atleast one cartridge that optionally contains support material. In someembodiments, the methods comprise the use of support material. Infurther embodiments, the tissues and organs fabricated by use of thedevices, systems, and methods described herein comprise support materialat the time of fabrication or thereafter.

In some embodiments, the support material is capable of excluding cellsgrowing or migrating into or adhering to it. In some embodiments, thesupport material is permeable for nutrient media.

In some embodiments, the viscosity of the support material ischangeable. In some embodiments, the viscosity of the support materialis changed by modifying the temperature of the support material. In someembodiments, the viscosity of the support material is changed bymodifying the pressure of the support material. In some embodiments, theviscosity of the support material is changed by modifying theconcentration of the support material. In some embodiments, theviscosity of the support material is changed by crosslinking (e.g., byuse of a chemical cross-linker), or photocrosslinking (e.g., usingultraviolet light exposure).

In some embodiments, the permeability of the support material ischangeable. In some embodiments, the permeability of the supportmaterial is modified by varying the temperature of the support materialor the temperature surrounding the support material. In someembodiments, the permeability of the support material is modified bycontacting the support material with an agent that modifiespermeability.

In some embodiments, the compliance (i.e., elasticity or hardness) ofthe support material is modified. In some embodiments, the compliance ofthe support material is modified by varying the temperature of thesupport material or the temperature surrounding the support material. Insome embodiments, the compliance of the support material is modified bycontacting the support material with an agent that modifies compliance.

Many support materials are suitable for use in the methods describedherein. In some embodiments, hydrogels are exemplary support materialspossessing one or more advantageous properties including: non-adherent,biocompatible, extrudable, bioprintable, non-cellular, and of suitablestrength. In some embodiments, suitable hydrogels are natural polymers.In one embodiment, the confinement material is comprised of NovoGel™. Infurther embodiments, suitable hydrogels include those derived fromsurfactant polyols (e.g., Pluronic F-127), collagen, hyaluronate,fibrin, alginate, agarose, chitosan, derivatives or combinationsthereof. In other embodiments, suitable hydrogels are syntheticpolymers. In further embodiments, suitable hydrogels include thosederived from poly(acrylic acid) and derivatives thereof, poly(ethyleneoxide) and copolymers thereof, poly(vinyl alcohol), polyphosphazene, andcombinations thereof. In various specific embodiments, the confinementmaterial is selected from: hydrogel, NovoGel™, agarose, alginate,gelatin, Matrigel™, hyaluronan, poloxamer, peptide hydrogel,poly(isopropyl n-polyacrylamide), polyethylene glycol diacrylate(PEG-DA), hydroxyethyl methacrylate, polydimethylsiloxane,polyacrylamide, poly(lactic acid), silicon, silk, or combinationsthereof.

In some embodiments, the support material contains cells prior to beingpresent in the bioprinter. In some embodiments, the support material isa hydrogel containing a suspension of cells. In some embodiments, thesupport material is a hydrogel containing a mixture of more than onecell type.

Exemplary Uses of Support Materials

In some embodiments, the support material is used as building units forconstructing a biological construct (e.g., cellular construct, tissue,organ, etc.). In further embodiments, the support material unit is usedto define and maintain the domains void of cellular material (i.e., theintermediate cellular units) of a desired construct. In someembodiments, the support material is capable of assuming any shape orsize.

For example, according to one embodiment, NovoGel™ solution (originallyin powder phase mixed with buffer and water) may be heated to reduce itsviscosity and taken up in a micropipette with a desired dimension (or ina chamber of a desired shape by negative displacement of a piston). TheNovoGel™ solution in the pipette (or the chamber) may be cooled to roomtemperature, for example by forced air on the exterior of the pipette(or the chamber) or plunging the micropipette into a container with coldliquid, so that it can solidify into a gel with the desired shape, i.e.,a support material. The resulting support material may be dispensed fromthe pipette or chamber during the construction of a particular cellularconstruct, tissue, or organ. See e.g., FIG. 4.

In some embodiments, the support material is used for increasing theviability of the engineered tissue or organ after bioprinting. Infurther embodiments, support material provides direct contact betweenthe tissue or organ and a nutrient medium through a temporary orsemi-permanent lattice of confinement material (e.g., support material).In some embodiments, the tissue is constrained in a porous or gappedmaterial. Direct access of at least some of the cells of the tissue ororgan to nutrients increases the viability of the tissue or organ.

In further embodiments, the methods disclosed herein comprise additionaland optional steps for increasing the viability of an engineered tissueor organ including: 1) optionally dispensing base layer of confinementmaterial (e.g., support material) prior to placing cohered multicellularaggregates; 2) optionally dispensing a perimeter of confinementmaterial; 3) bioprinting cells of the tissue within a defined geometry;and 4) dispensing elongate bodies (e.g., cylinders, ribbons, etc.) ofconfinement material overlaying the nascent tissue in a pattern thatintroduces gaps in the confinement material, such as a lattice, mesh, orgrid.

In some embodiments, the gaps overlaying pattern are distributeduniformly or substantially uniformly around the surface of the tissue ororgan. In other embodiments, the gaps are distributed non-uniformly,whereby the cells of the tissue or organ are exposed to nutrientsnon-uniformly. In non-uniform embodiments, the differential access tonutrients may be exploited to influence one or more properties of thetissue or organ. For instance, it may be desirable to have cells on onesurface of a bioprinted, cellular construct, tissue, or organproliferate faster than cells on another surface. In some embodiments,the exposure of various parts of the tissue or organ to nutrients can bechanged at various times to influence the development of the tissue ororgan toward a desired endpoint.

In some embodiments, the confinement material is removed at any suitabletime, including but not limited to, immediately after bioprinting (e.g.,within 10 minutes), after bioprinting (e.g., after 10 minutes), beforethe cells are substantially cohered to each other, after the cells aresubstantially cohered to each other, before the cells produce anextracellular matrix, after the cells produce an extracellular matrix,just prior to use, and the like. In various embodiments, confinementmaterial is removed by any suitable method. For example, in someembodiments, the confinement material is excised, pulled off the cells,digested, or dissolved.

Methods and Systems for Calibrating the Position of a BioprinterCartridge

Disclosed herein, in certain embodiments, are bioprinters forfabricating tissues and organs. In some embodiments, a cartridgeattached to the bioprinter comprises a bio-ink and/or a supportmaterial. In some embodiments, the bioprinter deposits the bio-ink orsupport material in a specific pattern and at specific positions inorder to form a specific tissue construct. In some embodiments, acartridge comprising bio-ink is disposable. In some embodiments, thecartridge is ejected from the bioprinter following extrusion,dispensing, or deposition of the contents. In some embodiments, a newcartridge is attached to the bioprinter.

In order to fabricate complex structures, the bioprinters disclosedherein dispense bio-ink and/or support material from a cartridge with asuitable repeatable accuracy. In various embodiments, suitablerepeatable accuracies include those of about ±5, 10, 20, 30, 40, or 50μm on any axis. In some embodiments, the bioprinters disclosed hereindispense bio-ink and/or support material from a cartridge with arepeatable accuracy of about ±20 μm. However, in some embodiments, dueto the removal and insertion of cartridges, the position of the printerhead (and thus the cartridges) with respect to the tissue constructvaries. Thus, there is a need for a method of precisely calibrating theposition of the printer head, cartridge, and dispensing orifice withrespect to the printer stage, receiving surface, tissue, or organ.

In some embodiments, the method of calibrating the position of a printerhead comprises use of at least one laser. In further embodiments, themethod of calibrating the position of a printer head comprises use of afirst and second laser.

In some embodiments, the method of calibrating the position of a printerhead comprises manual (e.g., visual) calibration.

In some embodiments, the method of calibrating the position of a printerhead comprises manual calibration and laser calibration.

In some embodiments, the position of the printer head is calibratedalong one axis, wherein the axis is selected from the x-axis, they-axis, and the z-axis. In some embodiments, the position of the printerhead is calibrated along two axes, wherein the axes are selected fromthe x-axis, the y-axis, and the z-axis. In some embodiments, theposition of the printer head is calibrated along three axes, wherein theaxes are selected from the x-axis, the y-axis, and the z-axis.

In some embodiments, calibration is made by use of at least one laser.In further embodiments, calibration is made by use of a first and asecond laser.

Method for Calibrating Using a Horizontal Laser

Disclosed herein, in certain embodiments, are methods of calibrating theposition of a printer head comprising a dispensing orifice. In someembodiments, a method disclosed herein further comprises activating alaser and generating at least one substantially stable and/orsubstantially stationary laser beam, and where said laser beam ishorizontal to the ground. See FIG. 1.

In some embodiments, the methods comprise, calibrating the position of aprinter head along at least one axis, wherein the axis is selected fromthe x-axis, y-axis, and z-axis. In some embodiments, the methodscomprise calibrating the position of the printer head along at least twoaxes, wherein the axis is selected from the x-axis, y-axis, and z-axis.In some embodiments, the methods comprise calibrating the position ofthe printer head along at least three axes, wherein the axis is selectedfrom the x-axis, y-axis, and z-axis. In some embodiments, the methodscomprise (a) calibrating the position of the printer head along they-axis; (b) calibrating the position of the printer head along thex-axis; and/or (c) calibrating the position of the printer head alongthe z-axis; wherein each axis corresponds to the axis of the same namein the Cartesian coordinate system. In some embodiments, calibration ismade by use of at least one laser. In some embodiments, calibration ismade by use of a first and a second laser.

In some embodiments, calibrating the position of a printer head alongthe y-axis comprises: (a) positioning the printer head so that theprinter head is (i) located in a first y octant and (ii) the dispensingorifice is below the upper threshold of the laser beam; (b) moving theprinter head towards the laser beam and stopping said movement as soonas the laser beam is interrupted by the printer head, wherein theposition at which the laser beam is interrupted by the printer head isthe first y position; (c) re-positioning the printer head so that theprinter head is located in the second y octant and the dispensingorifice is below the upper threshold of the laser beam; (d) moving theprinter head towards the laser beam and stopping said movement as soonas the laser beam is interrupted by the printer head, wherein theposition at which the laser beam is interrupted is the second yposition; (e) and calculating the mid-point between the first y positionand the second y position.

In some embodiments, calibrating the position of a printer head alongthe x-axis comprises: (a) positioning the printer head (i) at themid-point between the first y position and the second y position, and(ii) outside the sensor threshold of the laser; and (b) moving theprinter head towards the sensor threshold and stopping said movement assoon as the printer head contacts the sensor threshold; wherein theposition at which the printer head contacts the sensor increased by halfthe printer head width is the x position.

In some embodiments, calibrating the position of a printer head alongthe y-axis comprises: (a) positioning the printer head so that the laserbeam can measure the precise location of one side of the printer head;(b) positioning the printer head so that the laser beam can measure theprecise location of the opposing side of the printer head; (c) andcalculating the midpoint location of the printer head to be relative tothe laser location during each measurement and the measured distances.

In some embodiments, calibrating the position of a printer head alongthe x-axis comprises: (a) positioning the printer head so that the laserbeam can measure the precise location of one side of the printer head;(b) positioning the printer head so that the laser beam can measure theprecise location of the opposing side of the printer head; (c) andcalculating the midpoint location of the printer head to be relative tothe laser location during each measurement and the measured distances.

In some embodiments, calibrating the position of a printer head alongthe z-axis comprises: (a) positioning the printer head so that thedispensing orifice is located above the laser beam; and (b) moving theprinter head towards the laser beam and stopping said movement as soonas the laser beam is interrupted by the printer head, wherein theposition at which the laser beam is interrupted is the z position.

Method for Calibrating Using a Vertical Laser

Disclosed herein, in certain embodiments, are methods of calibrating theposition of a printer head comprising a dispensing orifice. In someembodiments, a method disclosed herein further comprises activating thelaser and generating at least one substantially stable and/orsubstantially stationary laser beam, and where said laser beam isvertical to the ground. See FIG. 2.

In some embodiments, the methods comprise, calibrating the position of aprinter head along at least one axis, wherein the axis is selected fromthe x-axis, y-axis, and z-axis. In some embodiments, the methodscomprise calibrating the position of a printer head along at least twoaxes, wherein the axis is selected from the x-axis, y-axis, and z-axis.In some embodiments, the methods comprise calibrating the position of aprinter head along at least three axes, wherein the axis is selectedfrom the x-axis, y-axis, and z-axis.

In some embodiments, the methods comprise (a) calibrating the positionof the printer head along the y-axis; (b) calibrating the position ofthe printer head along the x-axis; and (c) calibrating the position ofthe printer head along the z-axis; wherein each axis corresponds to theaxis of the same name in the Cartesian coordinate system.

In some embodiments, calibrating the position of a printer head alongthe y-axis comprises: (a) positioning the printer head so that theprinter head is (i) located in a first y octant and (ii) the dispensingorifice is outside the sensor threshold of the laser; (b) moving theprinter head towards the laser beam and stopping said movement as soonas the laser beam is interrupted by the printer head, wherein theposition at which the laser beam is interrupted by the printer head isthe first y position; (c) re-positioning the printer head so that theprinter head is located in the second y octant and the dispensingorifice is outside the sensor threshold of the laser; (d) moving theprinter head towards the laser beam and stopping said movement as soonas the laser beam is interrupted by the printer head, wherein theposition at which the laser beam is interrupted is the second yposition; (e) and calculating the mid-point between the first y positionand the second y position.

In some embodiments, calibrating the position of a printer head alongthe x-axis comprises: (a) positioning the printer head (i) at themid-point between the first y position and the second y position, and(ii) outside the sensor threshold of the laser; and (b) moving theprinter head towards the sensor threshold and stopping said movement assoon as the printer head contacts the sensor threshold; wherein theposition at which the printer head contacts the sensor increased by halfthe printer head width is the x position.

In some embodiments, calibrating the position of a printer head alongthe z-axis comprises: (a) positioning the printer head so that thedispensing orifice is located above the laser beam so that it is justoutside of the laser sensor range threshold; and (b) lowering theprinter head until the sensor threshold is reached, wherein the positionat which the laser sensor threshold is reached is the z position. Insome embodiments, steps (a) and (b) are repeated at multiple points ofthe printer head and measured heights are averaged to determine the zposition.

In some embodiments, calibrating the position of a printer head alongthe z-axis comprises: (a) positioning the printer head so that the laserbeam can measure the precise location of one or more points on thebottom of the printer head; (b) calculating the absolute or averagelocation of the printer head based on the laser position and knownmeasured distance.

Method for Calibrating Using a Vertical and Horizontal Laser

Disclosed herein, in certain embodiments, are methods of calibrating theposition of a printer head comprising a dispensing orifice, wherein theprinter head is attached to a bioprinter, comprising calibrating theposition of the printer head along at least one axis, wherein the axisis selected from the x-axis, y-axis, and z-axis. In some embodiments,the method comprises calibrating the position of a printer head along atleast two axes, wherein the axis is selected from the x-axis, y-axis,and z-axis. In some embodiments, the method comprises calibrating theposition of a printer head along at least three axes, wherein the axisis selected from the x-axis, y-axis, and z-axis.

In some embodiments, the methods comprise (a) calibrating the positionof the printer head along the y-axis; (b) calibrating the position ofthe printer head along the x-axis; and (c) calibrating the position ofthe printer head along the z-axis; wherein each axis corresponds to theaxis of the same name in the Cartesian coordinate system.

In some embodiments, calibration comprises use of a first laser and asecond laser. In some embodiments, the first laser is a vertical laserand the second laser is a horizontal laser.

System for Calibrating Using a Laser

Disclosed herein, in certain embodiments, are systems for calibratingthe position of a cartridge comprising a deposition orifice, wherein thecartridge is attached to a bioprinter, said system comprising: a meansfor calibrating the position of the cartridge along at least one axis,wherein the axis is selected from the y-axis, x-axis, and z-axis.

Also disclosed herein, in certain embodiments, are systems forcalibrating the position of a printer head comprising a dispensingorifice, wherein the printer head is attached to a bioprinter, saidsystem comprising: a means for calibrating the position of the printerhead along an x-axis; a means for calibrating the position of theprinter head along a y-axis; and a means for calibrating the position ofthe printer head along a z-axis.

In some embodiments, a system for calibrating the position of a printerhead comprises a means for calibrating the printer head along thex-axis, y-axis, and z-axis. In some embodiments, the means forcalibrating a printer head along the x-axis, y-axis, and z-axis is laseralignment, optical alignment, mechanical alignment, piezoelectricalignment, magnetic alignment, electrical field or capacitancealignment, ultrasound alignment, or a combination thereof.

In some embodiments, a system for calibrating the position of a printerhead comprises a means for calibrating the printer head along thex-axis, y-axis, and z-axis. In some embodiments, the means forcalibrating a printer head along the x-axis, y-axis, and z-axis is laseralignment. In some embodiments, the laser alignment means comprises atleast one laser. In some embodiments, the laser alignment meanscomprises a plurality of lasers.

In some embodiments, the laser alignment means it has any suitableaccuracy. In various embodiments, suitable accuracies include those ofabout ±5, 10, 20, 30, 40, or 50 μm on any axis. In some embodiments, thelaser alignment means is accurate to ±40 μm on the vertical axis and ±20μm on the horizontal axis.

In some embodiments, the laser path is uninterrupted between the lasersource and the measurement point. In some embodiments, the laser path isaltered by up to 179° by use of a reflective surface or optical lens. Insome embodiments, the laser path is altered by 90°. In some embodiments,a horizontal laser beam is used to measure in a vertical path bydeflection using a reflective surface. In some embodiments, a verticallaser beam is used to measure in a horizontal path by deflection using areflective surface.

EXAMPLES

The following specific examples are to be construed as merelyillustrative, and not limitative of the remainder of the disclosure inany way whatsoever. Without further elaboration, it is believed that oneskilled in the art can, based on the description herein, utilize thepresent invention to its fullest extent. All publications cited hereinare hereby incorporated by reference in their entirety. Referencethereto evidences the availability and public dissemination of suchinformation.

Example 1 HASMC-HAEC Mixed Cellular Cylinders

Cell Culture

Smooth Muscle Cells:

Primary human aortic smooth muscle cells (HASMC) were maintained andexpanded in low glucose Dulbecco's modified eagle medium (DMEM;Invitrogen Corp., Carlsbad, Calif.) supplemented with 10% fetal bovineserum (FBS), 100 U/ml Penicillin, 0.1 mg/ml streptomycin, 0.25 μg/ml ofamphotericin B, 0.01M of HEPES (all from Invitrogen Corp., Carlsbad,Calif.), 50 mg/L of proline, 50 mg/L of glycine, 20 mg/L of alanine, 50mg/L of ascorbic acid, and 3 μg/L of CuSO₄ (all from Sigma, St. Louis,Mo.) at 37° C. and 5% CO₂. Confluent cultures of HASMCs between passage4 and 8 were used in all studies.

Endothelial Cells:

Primary human aortic endothelial cells (HAEC) were maintained andexpanded in Medium 200 supplemented with 2% FBS, 1 μg/ml ofhydrocortisone, 10 ng/ml of human epidermal growth factor, 3 ng/ml ofbasic fibroblast growth factor, 10 μg/ml of heparin, 100 U/mlPenicillin, 0.1 mg/ml streptomycin, and 0.25 μg/ml of amphotericin B(all from Invitrogen Corp., Carlsbad, Calif.). The cells were grown ongelatin (from porcine serum; Sigma, St. Louis, Mo.) coated tissueculture treated flasks at 37° C. and 5% CO₂. Confluent cultures ofHAEC's between passage 4 and 8 were used in all studies.

NovoGel™ Mold

Preparation of 2% w/v NovoGel™ Solution:

1 g of low melting point NovoGel™ was dissolved in 50 ml of Dulbecco'sphosphate buffered saline (DPBS). Briefly, the DPBS and NovoGel™ wereheated to 85° C. on a hot plate with constant stirring until theNovoGel™ dissolved completely. NovoGel™ solution was sterilized by steamsterilization at 125° C. for 25 minutes. The NovoGel™ solution remainedin liquid phase as long as the temperature is maintained above 66.5° C.Below this temperature a phase transition occurs, the viscosity of theNovoGel™ solution increases and the NovoGel™ forms a solid gel.

Preparation of NovoGel™ Mold:

A NovoGel™ mold was fabricated for the incubation of cellular cylindersusing a Teflon® mold that fits a 10 cm Petri dish. Briefly, the Teflon®mold was pre-sterilized using 70% ethanol solution and subjecting themold to UV light for 45 minutes. The sterilized mold was placed on topof the 10 cm Petri dish (VWR International LLC, West Chester, Pa.) andsecurely attached. This assembly (Teflon® mold+Petri dish) wasmaintained vertically and 45 ml of pre-warmed, sterile 2% NovoGel™solution was poured in the space between the Teflon® mold and the Petridish. The assembly was then placed horizontally at room temperature for1 hour to allow complete gelation of the NovoGel™ After gelation, theTeflon® print was removed and the NovoGel™ mold was washed twice usingDPBS. 17.5 ml of HASMC culture medium was then added to the NovoGel™mold.

HASMC-HAEC Cylinders

Fabrication of HASMC-HAEC Mixed Cellular Cylinders:

To prepare mixed cellular cylinders HASMC and HAEC were individuallycollected and then mixed at pre-determined ratios. Briefly, the culturemedium was removed from confluent culture flasks and the cells werewashed with DPBS (1 ml/5 cm² of growth area). Cells were detached fromthe surface of the culture flasks by incubation in the presence oftrypsin (1 ml/15 cm² of growth area) for 10 minutes. HASMC were detachedusing 0.15% trypsin while HAEC were detached using 0.1% trypsin.Following the incubation appropriate culture medium was added to theflasks (2× volume with respect to trypsin volume). The cell suspensionwas centrifuged at 200 g for 6 minutes followed by complete removal ofsupernatant solution. Cell pellets were resuspended in respectiveculture medium and counted using a hemacytometer. Appropriate volumes ofHASMC and HAEC were combined to yield mixed cell suspensions containing5, 7.5, 10, 12.5, and 15% HAEC (as a % of total cell population). Themixed cell suspensions were centrifuged at 200 g for 5 minutes followedby complete removal of supernatant solution. Mixed cell pellets wereresuspended in 6 ml of HASMC culture medium and transferred to 20 mlglass vials, followed by incubation on an orbital shaker at 150 rpm for60 minutes, and at 37° C. and 5% CO₂. This allows the cells to aggregatewith one another and initiate cell-cell adhesions. Post-incubation, thecell suspension was transferred to a 15 ml centrifuge tube andcentrifuged at 200 g for 5 minutes. After removal of the supernatantmedium, the cell pellet was resuspended in 400 μl of HASMC culturemedium and pipetted up and down several times to ensure all cellclusters were broken. The cell suspension was transferred into a 0.5 mlmicrofuge tube placed inside a 15 ml centrifuge tube followed bycentrifugation at 2000 g for 4 minutes to form a highly dense andcompact cell pellet. The supernatant medium was aspirated and the cellswere transferred into capillary tubes (OD 1.0 mm, ID 0.5 mm, L 75 mm;Drummond Scientific Co., Broomall, Pa.) by aspiration so as to yieldcell cylinders 50 mm in length. The cell paste inside the capillarieswas incubated in HASMC medium for 20 minutes at 37° C. and 5% CO₂. Thecellular cylinders were then deposited from the capillary tubes into thegrooves of the NovoGel™ mold (covered with HASMC medium) using theplunger supplied with the capillaries. The cellular cylinders wereincubated for 24 and 48 hours at 37° C. and 5% CO₂.

Example 2 Multi-Layered Vascular Tubes

Cell Culture

Smooth Muscle Cells:

Primary human aortic smooth muscle cells (HASMC; GIBCO) were maintainedand expanded in low glucose dulbecco's modified eagle medium (DMEM)supplemented with 10% fetal bovine serum (FBS), 100 U/ml Penicillin, 0.1mg/ml streptomycin, 0.25 μg/ml of amphotericin B, 0.01M of HEPES (allfrom Invitrogen Corp., Carlsbad, Calif.), 50 mg/L of proline, 50 mg/L ofglycine, 20 mg/L of alanine, 50 mg/L of ascorbic acid, and 3 μg/L ofCuSO₄ (all from Sigma, St. Louis, Mo.) at 37° C. and 5% CO₂. Confluentcultures of HASMC between passage 4 and 8 were used in all studies.

Endothelial Cells:

Primary human aortic endothelial cells (HAEC) were maintained andexpanded in Medium 200 supplemented with 2% FBS, 1 μml/l ofhydrocortisone, 10 ng/ml of human epidermal growth factor, 3 ng/ml ofbasic fibroblast growth factor, 10 μg/ml of heparin, 100 U/mlPenicillin, 0.1 mg/ml streptomycin, and 0.25 μg/ml of amphotericin B(all from Invitrogen Corp., Carlsbad, Calif.). The cells were grown ongelatin (from porcine serum) coated tissue culture treated flasks at 37°C. and 5% CO₂. Confluent cultures of HAEC between passage 4 and 8 wereused in all studies.

Fibroblasts:

Primary human dermal fibroblasts (HDF) were maintained and expanded inMedium 106 supplemented with 2% FBS, 1 μg/ml of hydrocortisone, 10 ng/mlof human epidermal growth factor, 3 ng/ml of basic fibroblast growthfactor, 10 μg/ml of heparin, 100 U/ml Penicillin, and 0.1 mg/mlstreptomycin (all from Invitrogen Corp., Carlsbad, Calif.) at 37° C. and5% CO₂. Confluent cultures of HDF between passage 4 and 8 were used inall studies.

NovoGel™ Solutions and Mold

Preparation of 2% and 4% (w/v) NovoGel™ Solution:

1 g or 2 g (for 2% or 4% respectively) of low melting point NovoGel™(Ultrapure LMP) was dissolved in 50 ml of Dulbecco's phosphate bufferedsaline (DPBS). Briefly, the DPBS and NovoGel™ were heated to 85° C. on ahot plate with constant stirring until the NovoGel™ dissolvescompletely. NovoGel™ solution was sterilized by steam sterilization at125° C. for 25 minutes. The NovoGel™ solution remains in liquid phase aslong as the temperature is maintained above 66.5° C. Below thistemperature a phase transition occurs, the viscosity of the NovoGel™solution increases and the NovoGel™ forms a solid gel.

Preparation of NovoGel™ Mold:

A NovoGel™ mold was fabricated for the incubation of cellular cylindersusing a Teflon® mold that fit a 10 cm Petri dish. Briefly, the Teflon®mold was pre-sterilized using 70% ethanol solution and subjecting themold to UV light for 45 minutes. The sterilized mold was placed on topof the 10 cm Petri dish and securely attached. This assembly (Teflon®mold+Petri dish) was maintained vertically and 45 ml of pre-warmed,sterile 2% NovoGel™ solution was poured in the space between the Teflon®mold and the Petri dish. The assembly was then placed horizontally atroom temperature for 1 hour to allow complete gelation of the NovoGel™.After gelation, the Teflon® print was removed and the NovoGel™ mold waswashed twice using DPBS. Then, either 17.5 ml of HASMC culture mediumwas added to the NovoGel™ mold for incubating HASMC-HAEC mixed cellcylinders or 17.5 ml of HDF culture medium is added to the NovoGel™ moldfor incubating HDF cell cylinders.

Cellular Cylinders

Fabrication of HASMC-HAEC Mixed Cellular Cylinders:

To prepare mixed cellular cylinders HASMC and HAEC were individuallycollected and then mixed at pre-determined ratios. Briefly, the culturemedium was removed from confluent culture flasks and the cells werewashed with DPBS (1 ml/5 cm² of growth area). Cells were detached fromthe surface of the culture flasks by incubation in the presence oftrypsin (1 ml/15 cm² of growth area) for 10 minutes. HASMC were detachedusing 0.15% trypsin while HAEC were detached using 0.1% trypsin.Following the incubation appropriate culture medium was added to theflasks (2× volume with respect to trypsin volume). The cell suspensionwas centrifuged at 200 g for 6 minutes followed by complete removal ofsupernatant solution. Cell pellets were resuspended in respectiveculture medium and counted using a hemacytometer. Appropriate volumes ofHASMC and HAEC were combined to yield a mixed cell suspension containing15% HAEC and remainder 85% HASMC (as a percentage of total cellpopulation). The mixed cell suspension was centrifuged at 200 g for 5minutes followed by complete removal of supernatant solution. Mixed cellpellets were resuspended in 6 ml of HASMC culture medium and transferredto 20 ml glass vials, followed by incubation on an orbital shaker at 150rpm for 60 minutes, and at 37° C. and 5% CO₂. This allows the cells toaggregate with one another and initiate cell-cell adhesions.Post-incubation, the cell suspension was transferred to a 15 mlcentrifuge tube and centrifuged at 200 g for 5 mins. After removal ofthe supernatant medium, the cell pellet was resuspended in 400 μl ofHASMC culture medium and pipetted up and down several times to ensureall cell clusters were broken. The cell suspension was transferred intoa 0.5 ml microfuge tube placed inside a 15 ml centrifuge tube followedby centrifugation at 2000 g for 4 minutes to form a highly dense andcompact cell pellet. The supernatant medium was aspirated and the cellswere transferred into capillary tubes (OD 1.0 mm, ID 0.5 mm, L 75 mm) byaspiration so as to yield cell cylinders 50 mm in length. The cell pasteinside the capillaries was incubated in HASMC medium for 20 minutes at37° C. and 5% CO₂. The cellular cylinders were then deposited from thecapillary tubes into the grooves of the NovoGel™ mold (covered withHASMC medium) using the plunger supplied with the capillaries. Thecellular cylinders were incubated for 24 hours at 37° C. and 5% CO₂.

Fabrication of HDF Cell Cylinders:

HDF cylinders were prepared using a method similar to preparingHASMC-HAEC mixed cellular cylinders. Briefly, the culture medium wasremoved from confluent HDF culture flasks and the cells were washed withDPBS (1 ml/5 cm² of growth area). Cells were detached from the surfaceof the culture flasks by incubation in the presence of trypsin (0.1%; 1ml/15 cm² of growth area) for 10 minutes. Following the incubation HDFculture medium was added to the flasks (2× volume with respect totrypsin volume). The cell suspension was centrifuged at 200 g for 6minutes followed by complete removal of supernatant solution. Cellpellets were resuspended in 6 ml of HDF culture medium and transferredto 20 ml glass vials, followed by incubation on an orbital shaker at 150rpm for 75 minutes, and at 37° C. and 5% CO₂. Post-incubation, the cellsuspension was transferred to a 15 ml centrifuge tube and centrifuged at200 g for 5 minutes. After removal of the supernatant medium, the cellpellet was resuspended in 400 μl of HDF culture medium and pipetted upand down several times to ensure all cell clusters were broken. The cellsuspension was transferred into a 0.5 ml microfuge tube placed inside a15 ml centrifuge tube followed by centrifugation at 2000 g for 4 minutesto form a highly dense and compact cell pellet. The supernatant mediumwas aspirated and the cells were transferred into capillary tubes (OD1.0 mm, ID 0.5 mm, L 75 mm) by aspiration so as to yield cell cylinders50 mm in length. The cell paste inside the capillaries were incubated inHDF culture medium for 20 minutes at 37° C. and 5% CO₂. The cellularcylinders were then deposited from the capillary tubes into the groovesof the NovoGel™ mold (covered with HDF medium). The cellular cylinderswere incubated for 24 hours at 37° C. and 5% CO₂.

Fabrication of Multi-Layered Vascular Tubes

Preparation of NovoGel™ Base Plate:

A NovoGel™ base plate was fabricated by dispensing 10 ml of pre-warmed(>40° C.) NovoGel™ (2% w/v) into a 10 cm Petri dish. Immediately afterdispensing, the NovoGel™ was evenly spread so as to cover the entirebase of the dish and form a uniform layer. The Petri dish was incubatedat room temperature for 20 minutes to allow the NovoGel™ to gelcompletely.

Multi-Layered Vascular Tube:

Vascular tubes consisting of an outer layer of HDF and an inner layer ofHASMC-HAEC were fabricated utilizing HDF cylinders, and HASMC-HAEC mixedcell cylinders. A geometrical arrangement as shown in FIG. 4 wasutilized. Briefly, at the end of the 24-hour incubation period matureHDF and HASMC-HAEC cylinders were aspirated back into the capillarytubes and placed in appropriate culture medium until further use. Thesupport structure consisting of NovoGel™ rods was prepared as follows:Pre-warmed 2% NovoGel™ was aspirated into the capillary tubes (L=50 mm)and rapidly cooled in cold PBS solution (4° C.). The 5 cm long gelledNovoGel™ cylinder was deposited from the capillary (using the plunger)and laid down straight on the NovoGel™ base plate. A second NovoGel™cylinder was adjoined to the first one and the process was repeateduntil 10 NovoGel™ cylinders were deposited to form the first layer. Atthis point 20 μl of PBS was dispensed above the NovoGel™ cylinders tokeep them wet. Further six NovoGel™ cylinders were deposited on top oflayer 1 at positions as shown in FIG. 4 (layer 2). Three HDF cylinderswere then deposited at positions 4, 5 and 6 to complete layer 2. Afterdispensing each HDF cylinder 40 μl of HDF culture medium was dispensedon top of the deposited cylinder to assist the deposition of thesubsequent cylinder as well as to prevent dehydration of the cellularcylinders. Next NovoGel™ cylinders for layer 3 were deposited followedby HDF cylinders at positions 3 and 6. Following rewetting of thestructure with HDF culture medium, HASMC-HAEC mixed cylinders were laiddown in positions 4 and 5. Subsequently, 40 μl of HASMC medium and 40 μlof HDF medium were dispensed on top of the cell cylinders. Layer 4 wascompleted by depositing NovoGel™ cylinders at positions 1 and 7, HDFcylinders at positions 2 and 6, HASMC-HAEC mixed cylinders at positions3 and 5, and finally a 4% NovoGel™ cylinder at position 4. Layers 5, 6and 7 were completed similarly by laying down NovoGel™ cylindersfollowed by HDF cylinders and finally HASMC-HAEC cylinders at positionsshown in FIG. 4. Once the entire construct was completed 0.5 ml of warmNovoGel™ was dispensed over each end of the construct and allowed to gelat room temperature for 5 minutes. Following gelation of that NovoGel™,30 ml of HASMC medium was added to the Petri dish (to ensure the entireconstruct was completely submerged). The construct was incubated for 24hours at 37° C. and 5% CO₂ to allow for fusion between the cellularcylinders.

At the end of 24 hours, the surrounding NovoGel™ support structure wasremoved from the fused multi-layered vascular tube.

Example 3 Bioprinter

A bioprinter was assembled. The bioprinter contained a printer headhaving a collet chuck grip for holding a cartridge, and a piston fordispensing the contents of the cartridge. The cartridges used were glassmicrocapillary tubes having a length of 75-85 mm. A new capillary tubewas loaded each time bio-ink or support material was required.

In order to print structures, a dispense position repeatability of ±20μm was required for the duration of the printing process, i.e., when newcapillaries were loaded into the printer head. In order to maintainrepeatability of all loaded capillary tubes relative to the same pointin the x-, y-, and z-directions, the bioprinter contained a lasercalibration system for calibrating the position of the microcapillarytube. The laser calibration system calibrated the position of allcapillary tips to a common reference location. All printing moves weremade relative to this reference position.

All three axes (x-, y-, and z-axes) were calibrated through usage of asingle laser distance measurement sensor. The system consisted of alaser sensor and a laser beam. The sensor threshold was the maximumsensing distance of the laser sensor. The sensor was configured toignore all signals further away than a pre-defined threshold. The sensorused triangulation to determine distance to the object (the capillarytip). The laser sensor was orientated with the beam aimed vertically up(+z-axis).

Vertical Laser Calibration

For Calibration in the X-Axis:

The capillary tip was moved in the range of the laser sensor, with thetip to the left (−x) of the laser beam. The capillary was moved to inthe +x direction until the sensor detected the capillary edge, and thisposition was recorded. The above steps were repeated from the oppositeside (i.e., the tip was positioned at the right (+x) of the laser beamand moved in the −x direction until the sensor detected the capillaryedge). The positions from both steps were averaged to calculate themid-point of the capillary. Optionally, the above process was repeatedfor different y-positions and the calculated mid-points were averaged.

For Calibration in the Y-Axis:

The above procedure (for the x-axis) was repeated for the y-axis.

For Calibration in the Z-Axis:

The capillary tip was moved to above the sensor beam so that the beanhit the bottom surface of the capillary, and the tip was just outside ofthe sensor range threshold. The capillary was lowered until the sensorthreshold was reached, and that position was recorded as the z-position.Optionally, the above steps were repeated at multiple points on thecapillary tip surface and measured heights were averaged.

Horizontal Laser Calibration

For Calibration in the Y-Axis:

The capillary was moved so that the tip was just below the laser beamheight, and the capillary was off to one side (in the y-direction). Thecapillary was moved in the y-direction towards the laser. The capillarywas stopped when the laser sensor detected the beam reflected off thecapillary, and this position was recorded. The above steps were repeatedwith the capillary off to the other side of the laser, and moved in the−y direction). The mid-point from the above steps was recorded as they-position.

For Calibration in the X-Axis:

Using the results of the calibration in the y-axis, the y-axis was movedso that the laser was centered on the capillary. The capillary was movedpast the sensor threshold and moved towards the sensor. The capillarywas stopped as soon as the capillary crossed the sensor threshold andthe sensor output changed. This position, plus ½ the capillary width(from the y-calibration) was recorded as the x-position.

For Calibration in the Z-Axis:

The capillary was moved up from the x-position until it was clear of thelaser beam. The capillary tip was moved down towards the laser beam, andstopped as soon as the laser beam was interrupted (using the sameprocess as for the y-axis). This position was recorded as thez-position.

Capillary Priming

Before printing from a capillary, the bio-ink or support material insidethe capillary was primed so that the bio-ink or support material wouldbegin printing at the very tip of the capillary. The calibration laserwas used to prime the capillary. The capillary tip was moved just abovethe laser beam, with the beam centered in the y-axis. The tip wasbetween 20-100 μm above the laser beam. The dispensing piston in theprinter head was driven down until the bio-ink or support materialstarted to dispense out of the capillary tip and interrupted the laserbeam. The dispensed bio-ink or support material was aspirated back intothe capillary tube by driving the piston in the reverse direction(20-100 μm). The capillary was then primed and ready to dispense.

NovoGel™ Capillary Cleaning

NovoGel™ was used as a support material. In order to remove excessNovoGel™ sticking to the outside surface of the capillary tube and toavoid the excess NovoGel™ from affecting print quality, the excessNovoGel™ was removed. A wiping feature was integrated into a bulkNovoGel™ vessel. A bulk NovoGel™ vessel was fitted with a standardmedical vial with an open cap for a septum to be attached. A septum wasconfigured with a cross cut in the center of 1-2 mm thick silicone. Bydipping the capillary into the bulk NovoGel™ vessel through the septumand aspirating NovoGel™, excess NovoGel™ was wiped from the capillary asit exited the vessel, and remained in the bulk vessel.

Printing of a Vascular Structure

The bioprinter and cartridge was assembled as above. The bioprinter hada stage having a Petri dish for receiving structures generated by thebioprinter. The Petri dish was coated with NovoGel™.

A two dimensional representation (see e.g., FIG. 4) of a vascularstructure was inputted by a user into a software program into a computerwhich was connected to the bioprinter. The two dimensionalrepresentation of the vascular structure consisted of rods of HASMC-HAECmixed cellular cylinders, HDF cylinders, and NovoGel™ rods defining thevoids of the vascular structure and surrounding the vascular structure.HASMC-HAEC mixed cellular cylinders and HDF cellular cylinders wereprepared as in Example 1, and aspirated into capillary tubes forinsertion into the collet chuck of the printer head. Alternatively,capillary tubes were loaded into the printer head and dipped into thebulk NovoGel™ vessel and NovoGel™ was aspirated into the capillary tube.The capillary tubes were calibrated using the vertical laser calibrationsystem.

When the commands from the software program were provided to thebioprinter, the bioprinter would print the three-dimensional structure,alternating between HASMC-HAEC rods, HDF rods and NovoGel™ rods, ontothe Petri dish, in predetermined locations. See Example 2. After eachrod was laid down on the Petri dish, the rod was wetted with a smallamount of culture medium. Once the entire construct was completed warmNovoGel™ was dispensed over each end of the construct and allowed to gelat room temperature, and cell culture medium was added to the Petri dishto submerge the entire construct. The construct was then incubated at37° C. and 5% CO₂ to allow for fusion between the cellular cylinders. Atthe end of the incubation time, the surrounding NovoGel™ supportstructure was removed from the fused multi-layered vascular tube.

While the invention has been described in connection with specificembodiments thereof, it will be understood that the inventivemethodology is capable of further modifications. This patent applicationis intended to cover any variations, uses, or adaptations of theinvention following, in general, the principles of the invention andincluding such departures from the present disclosure as come withinknown or customary practice within the art to which the inventionpertains and as may be applied to the essential features herein beforeset forth and as follows in scope of the appended claims.

What is claimed is:
 1. A three-dimensional bioprinter comprising: a. oneor more printer heads, wherein a printer head comprises a means forreceiving and holding at least one cartridge, wherein a cartridgecomprises at least one deposition orifice and contents comprising abio-ink, wherein the bio-ink comprises solid or semi-solid aggregates ofliving cells; b. a means for calibrating a position in space of the atleast one cartridge or the at least one deposition orifice utilizing alaser and a laser detector; c. a means for extruding the contents of theat least one cartridge by application of pressure, utilizing a piston;d. a receiving surface for receiving the extruded contents of the atleast one cartridge; and e. a programmable computer processorcommunicatively connected to the means for calibrating and the means forextruding, the programmable computer processor for positioning the oneor more printer heads in three dimensions and regulating the extrusionof the contents of the at least one cartridge.
 2. The bioprinter ofclaim 1, further comprising a means for applying a wetting agent,utilizing a sprayer or a pipette, to one or more of: a. the receivingsurface; b. the at least one deposition orifice; c. the bio-ink; and d.the extruded contents of the at least one cartridge; wherein the wettingagent is applied at one or more time points selected from: before thebio-ink is extruded by the bioprinter, substantially concurrently withextruding, and after the bio-ink is extruded by the bioprinter.
 3. Thebioprinter of claim 1, wherein the means for calibrating the position ofthe at least one cartridge or the at least one deposition orificecalibrates along an x-axis, a y-axis, and a z-axis, utilizing one ormore lasers and the laser detector.
 4. The bioprinter of claim 1,wherein the computer processor is programmable by: a. a graphical userinterface that is capable of receiving input of a visual representationof a desired tissue construct; and b. generating a series of commands,wherein the commands are based on the visual representation inputted viathe graphical user interface.
 5. The bioprinter of claim 4, wherein thevisual representation comprises identification of one or more elementsof a desired tissue construct as one or more of: a. the bio-ink; and b.a specific composition of the bio-ink.
 6. The bioprinter of claim 1,wherein the one or more printer heads, the at least one cartridge, orthe at least one deposition orifice are moved relative to the receivingsurface, utilizing the laser and the laser detector, to produce adesired tissue construct of a particular three-dimensional geometryinputted via the graphical user interface in the extruded contents ofthe at least one cartridge.
 7. The bioprinter of claim 1, wherein thereceiving surface is moved relative to the one or more printer heads,the at least one cartridge, or the at least one deposition orifice,utilizing the laser and the laser detector, to produce a desired tissueconstruct of a particular three dimensional geometry inputted via thegraphical user interface, in the extruded contents of the at least onecartridge.
 8. The bioprinter of claim 1, wherein the solid or semi-solidaggregates of living cells are substantially elongate.
 9. The bioprinterof claim 8, wherein the solid or semi-solid aggregates of living cellsare substantially in the form of cylinders or ribbons.
 10. Thebioprinter of claim 1, wherein the solid or semi-solid aggregates ofliving cells cellular aggregates are substantially spheroid.